Process For Producing Glycolic Acid From Formaldehyde and Hydrogen Cyanide

ABSTRACT

A process is provided for producing glycolic acid from formaldehyde and hydrogen cyanide. More specifically, heat-treated formaldehyde and hydrogen cyanide are reacted to produce glycolonitrile having low concentrations of impurities. The glycolonitrile is subsequently converted to an aqueous solution of ammonium glycolate using an enzyme catalyst having nitrilase activity derived from  Acidovorax facilis  72W (ATCC 57746). Glycolic acid is recovered in the form of the acid or salt from the aqueous ammonium glycolate solution using a variety of methods described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/314,905, filed on Dec. 21, 2005, which claims the benefit of U.S.Provisional Application Nos. 60/638,168; 60/638,148; 60/638,176;60/638,127; 60/638,128; and 60/638,126; each filed Dec. 22, 2004. Eachof these applications is incorporated herein by reference.

TECHNICAL FIELD

This invention relates to the field of organic acid synthesis, molecularbiology, and microbiology. More specifically, a process for theproduction of glycolic acid from formaldehyde and hydrogen cyanide isprovided using an enzyme catalyst having nitrilase activity.

BACKGROUND

Glycolic acid (HOCH₂COOH; CAS Registry Number is 79-14-1) is thesimplest member of the α-hydroxy acid family of carboxylic acids. Itsproperties make it ideal for a broad spectrum of consumer and industrialapplications, including use in water well rehabilitation, the leatherindustry, the oil and gas industry, the laundry and textile industry, asa monomer in the preparation of polyglycolic acid (PGA), and as acomponent in personal care products. Glycolic acid also is a principleingredient for cleaners in a variety of industries (dairy and foodprocessing equipment cleaners, household and institutional cleaners,industrial cleaners [for transportation equipment, masonry, printedcircuit boards, stainless steel boiler and process equipment, coolingtower/heat exchangers], and metals processing [for metal pickling,copper brightening, etching, electroplating, electropolishing]).Recently, it has been reported that polyglycolic acid is useful as a gasbarrier material (i.e., exhibits high oxygen barrier characteristics)for packing foods and carbonated drinks (WO 2005/106005 A1). However,traditional chemical synthesis of glycolic acid produces a significantamount of impurities that must be removed prior to use in preparingpolyglycolic acid for gas barrier materials. New technology tocommercially produce glycolic acid, especially one that producesglycolic acid in high purity and at low cost, would be eagerly receivedby industry.

Microbial catalysts can hydrolyze a nitrile (e.g., glycolonitrile)directly to the corresponding carboxylic acids (e.g., glycolic acid)using a nitrilase (EC 3.5.5.7), where there is no intermediateproduction of the corresponding amide (Equation 1), or by a combinationof nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) enzymes,where a nitrile hydratase (NHase) initially converts a nitrile to anamide, and then the amide is subsequently converted by the amidase tothe corresponding carboxylic acid (Equation 2):

Enzymatic synthesis of glycolic acid requires a substantially pure formof glycolonitrile. Methods to synthesize glycolonitrile by reactingaqueous solutions of formaldehyde and hydrogen cyanide have previouslybeen reported (U.S. Pat. No. 2,175,805; U.S. Pat. No. 2,890,238; andU.S. Pat. No. 5,187,301; Equation 3).

HCN+HCHO→HOCH₂CN  (3)

However, these methods typically result in an aqueous glycolonitrilereaction product that requires significant purification (e.g.,distillative purification) as many of the impurities and/or byproductsof the reaction (including excess reactive formaldehyde) may interferewith the enzymatic conversion of glycolonitrile to glycolic acid,including catalyst inactivation. Inactivation of the enzyme catalystdecreases the overall productivity of the catalyst (i.e., total grams ofglycolic acid formed per gram of catalyst), adding a significant cost tothe overall process, which may make enzymatic production methodseconomically non-viable when compared to chemical methods of production.As such, reaction conditions that yield glycolonitrile with fewerimpurities are needed, especially those that address the amount of freeformaldehyde in the reaction product. The glycolonitrile synthesisconditions should 1) increase overall glycolonitrile yield, 2) minimizeunwanted impurities and/or byproducts, and 3) decrease the cost to makea glycolonitrile preparation suitable for enzymatic synthesis.

Various methods are known for preparing α-hydroxy acids using thecorresponding α-hydroxy nitrile as the starting material and amicroorganism as the catalyst. Examples of α-hydroxy acids producedinclude: glycolic acid, lactic acid, 2-hydroxyisobutyric acid,2-hydroxy-2-phenyl propionic acid, mandelic acid,2-hydroxy-3,3-dimethyl-4-butyrolactone, and 4-methylthiobutyric acid.These products are synthesized using microorganisms, such as thosebelonging to the genera Nocardia, Bacillus, Brevibacterium,Aureobacterium, Pseudomonas, Caseobacter, Alcaligenes, Acinetobacter,Enterobacter, Arthrobacter, Escherichia, Micrococcus, Streptomyces,Flavobacterium, Aeromonas, Mycoplana, Cellulomonas, Erwinia, Candida,Bacteridium, Aspergillus, Penicillium, Cochliobolus, Fusarium,Rhodopseudomonas, Rhodococcus, Corynebacterium, Microbacterium,Obsumbacterium and Gordona. (JP-A-4-99495, JP-A-4-99496 andJP-A-4-218385 corresponding to U.S. Pat. No. 5,223,416; JP-A-4-99497corresponding to U.S. Pat. No. 5,234,826; JP-A-5-95795 corresponding toU.S. Pat. No. 5,296,373; JP-A-5-21987; JP-A-5-192189 corresponding toU.S. Pat. No. 5,326,702; JP-A-6-237789 corresponding to EP-A-0610048;JP-A-6-284899 corresponding to EP-A-0610049; JP-A-7-213296 correspondingto U.S. Pat. No. 5,508,181).

However, most known methods for preparing α-hydroxy acids from thecorresponding α-hydroxy nitriles as mentioned above do not produce andaccumulate a product at a sufficiently high concentration to meetcommercial needs. This is frequently a result of enzyme inactivationearly in the reaction period. U.S. Pat. No. 5,756,306 teaches that “Whenan α-hydroxy nitrile is enzymatically hydrolyzed or hydrated usingnitrilase or nitrile hydratase to produce an α-hydroxy acid or α-hydroxyamide, a problem occurs in that the enzyme is inactivated within a shortperiod of time. It is therefore difficult to obtain the α-hydroxy acidor α-hydroxy amide in high concentration and high yield.” (col. 1, lines49-54). Maintaining the aldehyde concentration (formed by thedisassociation of α-hydroxy nitrile to aldehyde and hydrogen cyanide)and/or the α-hydroxy nitrile concentration in the reaction mixturewithin a specified range is one method to avoid this problem.

U.S. Pat. No. 5,508,181 addresses further difficulties relating to rapidenzyme inactivation. Specifically, U.S. Pat. No. 5,508,181 mentions thatα-hydroxy nitrile compounds partially disassociate into thecorresponding aldehydes, according to the disassociation equilibrium.These aldehydes inactivate the enzyme within a short period of time bybinding to the protein, thus making it difficult to obtain α-hydroxyacid or α-hydroxy amide in a high concentration with high productivityfrom α-hydroxy nitriles (col. 2, lines 16-29). As a solution to preventenzyme inactivation due to accumulation of aldehydes, phosphate orhypophosphite ions were added to the reaction mixture. U.S. Pat. No.5,326,702 uses sulfite, disulfite, or dithionite ions to sequesteraldehyde and prevent enzyme inactivation. However, the concentration ofα-hydroxy acid produced and accumulated even by using such additives asdescribed above is not great.

U.S. Pat. No. 6,037,155 teaches that low accumulation of α-hydroxy acidproduct is related to enzyme inactivation within a short time due to thedisassociated-aldehyde accumulation. These inventors suggest thatenzymatic activity is inhibited in the presence of hydrogen cyanide(Asano et al., Agricultural Biological Chemistry, Vol. 46, pages1165-1174 (1982)) generated in the partial disassociation of theα-hydroxy nitrile in water together with the corresponding aldehyde orketone (Mowry, David T., Chemical Reviews, Vol. 42, pages 189-283(1948)). The inventors solved the problem of aldehyde-induced enzymeinactivation by using microorganisms whose enzyme activity could beimproved by adding a cyanide substance to the reaction mixture. Theaddition of a cyanide substance limited the disassociation of α-hydroxynitrile to aldehyde and hydrogen cyanide.

With specific respect to the production of glycolic acid, glycolonitrileis known to reversibly disassociate to hydrogen cyanide andformaldehyde, either of which may be involved in enzyme inactivation.U.S. Pat. No. 3,940,316 describes a process for preparing an organicacid from the corresponding nitrile using bacteria with “nitrilasic”activity, and lists glycolonitrile as a substrate. In particular, thispatent describes the use of Bacillus, Bacteridium, Micrococcus, andBrevibacterium for this purpose. Though described as having nitrilasicactivity, Brevibacterium R312 is the only strain used in all of the U.S.Pat. No. 3,940,316 examples. Brevibacterium R312 is known to havenitrile hydratase and amidase activities, but no nitrilase activity(Tourneix et al., Antonie van Leeuwenhoek, 52:173-182 (1986)).

A method for preparing lactic acid, glycolic acid, and2-hydroxyisobutyric acid by using a microorganism belonging toCorynebacterium spp. is disclosed in Japanese Patent Laid-open No. Sho61-56086. JP 09028390 discloses a method for manufacturing glycolic acidfrom glycolonitrile by the action of Rhodococcus or Gordona hydrolase.Selectivity for glycolic acid is reported as almost 100%, withoutformation of glycolic acid amide. U.S. Pat. No. 6,037,155 disclosesexamples of methods for producing α-hydroxy acids from α-hydroxynitriles, including glycolic acid. This disclosure acknowledges that notall microbial catalysts can produce high concentrations of glycolic aciddue to the aforementioned problems and instructs that screening studiesmust be conducted in order to find industrially advantageousmicroorganisms. U.S. Pat. No. 6,037,155 specifically identifiesVariovorax spp. and Arthrobacter spp. microorganisms that are resistantto the suppressing effect of α-hydroxy nitrile or α-hydroxy acid, havedurable activity, and can produce the desired product at highconcentration.

Acidovorax facilis 72W (ATCC 55746) is characterized by aliphaticnitrilase (EC 3.5.5.7) activity, as well as a combination of nitrilehydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. The geneencoding the A. facilis 72W (ATCC 55746) nitrilase has been cloned andrecombinantly expressed (WO 01/75077 corresponding to U.S. Pat. No.6,870,038) and Chauhan et al., Appl Microbiol Biotechnol, 61:118-122(2003)). The A. facilis 72W nitrilase converts α-hydroxynitriles to thecorresponding α-hydroxycarboxylic acids in high yield (U.S. Pat. No.6,383,786), including glycolic acid (U.S. Pat. No. 6,416,980). However,enzyme catalysts having improved nitrilase activity relative to the A.facilis 72W nitrilase when converting glycolonitrile to glycolic acid inhigh yield at up to 100% conversion would be very useful in reducing thecost of manufacturing glycolic acid.

A process to economically produce glycolic acid using an enzyme catalystrequires 1) a source of high purity glycolonitrile, 2) the use of anenzyme catalyst that can convert glycolonitrile to glycolic acid in highconcentrations with high purity, and 3) a method recovering the glycolicacid produced. In one embodiment, the process includes use of an enzymecatalyst having high catalyst productivity (kg glycolic acid/kg enzymecatalyst) and volumetric productivity (grams of glycolic acid/L/h). Theenzyme catalyst may be employed in multiple consecutive batch reactions,or in a continuous reaction that employs constant addition ofglycolonitrile and removal of glycolic acid; in either mode ofoperation, the catalyst activity and lifetime should be such that a highvolumetric productivity and catalyst productivity are obtained, and inthe case of batch reactions, the catalyst must be utilized in multiplereaction cycles without significant loss in enzyme activity betweenconsecutive batch reactions. Nitrilases having improved activity forglycolonitrile hydrolysis can provide improvements in volumetricproductivity. Given the fact that the inactivating effect of freeformaldehyde (and possibly other impurities) in the glycolonitrilereaction mixture will negatively affect all nitrilase catalysts tovarying extents, improvements that stabilize enzyme activity underreaction conditions for hydrolysis of glycolonitrile (resulting in arelative increase in catalyst productivity) are also needed.

Enzymatic conversion of glycolonitrile to glycolic acid using an enzymecatalyst normally results in the production of an aqueous solutioncomprising mostly ammonium glycolate (i.e., reactions are typically runat a pH of about 6 to about 9). Various methods can be used to obtainglycolic acid from aqueous solutions of ammonium glycolate including,but not limited to ion exchange (anionic and/or cationic),electrodialysis, reactive solvent extraction, polymerization, thermaldecomposition (salt cracking), alcoholysis, and combinations thereof.

The problem to be solved is to provide a process to produce glycolicacid (in the form of the salt of acid) in high yield and with highpurity. In one embodiment, the desired process should include 1)preparation of an aqueous solution comprising glycolonitrile fromformaldehyde and hydrogen cyanide suitable for enzymatic conversion toammonium glycolate (i.e., “high purity” glycolonitrile), 2) use of anenzyme catalyst having nitrilase activity the hydrolyzes the high purityglycolonitrile into ammonium glycolate, and 3) a method to obtain highpurity glycolic acid from the ammonium glycolate. In another embodiment,the process includes use of an enzyme catalyst having improved nitrilaseactivity (thereby increasing volumetric productivity) relative to thenitrilase activity of the Acidovorax facilis 72W nitrilase, and reactionconditions that improve catalyst stability and productivity.

SUMMARY

The present problem has been solved by providing a process to produceglycolic acid from formaldehyde and hydrogen cyanide comprising:

-   -   a) providing an aqueous formaldehyde feed stream heated to a        temperature of about 90° C. to about 150° C. for a determinable        period of time;    -   b) contacting the heated aqueous formaldehyde feed stream of (a)        with hydrogen cyanide at a temperature suitable for        glycolonitrile synthesis, whereby glycolonitrile is produced;    -   c) contacting the glycolonitrile produced in step (b) in a        suitable aqueous enzymatic reaction mixture with an enzyme        catalyst comprising a polypeptide having nitrilase activity,        said polypeptide having an amino acid sequence of SEQ ID NO: 6        with at least one amino acid substitution selected from the        group consisting of:        -   (1) a substitution at amino acid residue 168 with lysine,            methionine, threonine or valine; and        -   (2) a substitution at amino acid residue 201 with glutamine,            glycine, histidine, lysine, asparagine, serine, alanine,            cysteine, or threonine; whereby glycolic acid is produced in            the form of a salt or acid; wherein said enzyme catalyst            provides at least a 1.5-fold increase in nitrilase activity            relative to the nitrilase activity of the Acidovorax facilis            72W nitrilase catalyst when converting glycolonitrile to            glycolic acid under identical reaction conditions; and    -   d) recovering the glycolic acid produced in (c) in the form of a        salt or acid.

In one embodiment, the methods of recovering the glycolic acid in stepd) include, but are not limited to ion exchange, electrodialysis,reactive solvent extraction, thermal decomposition, alcoholysis,polymerization, and combinations thereof.

The high purity glycolonitrile produced using the present processenables production of high purity glycolic acid when using an enzymecatalyst comprising a polypeptide having nitrilase activity (i.e, anitrilase) in combination with ion exchange. As such, a process forproducing glycolic acid from formaldehyde and hydrogen cyanide isprovided comprising:

-   -   (a) providing an aqueous formaldehyde feed stream that is heated        to a temperature of about 90° C. to about 150° C. for a        determinable period of time;    -   (b) contacting the heated aqueous feed stream of (a) with        hydrogen cyanide at a temperature suitable for glycolonitrile        synthesis, whereby glycolonitrile is produced;    -   (c) contacting the glycolonitrile of step (b) in a suitable        aqueous reaction mixture with an enzyme catalyst comprising a        polypeptide having nitrilase activity, whereby glycolic acid is        produced; and    -   (d) recovering the glycolic acid produced in (c) by ion        exchange;

wherein said glycolic acid has a purity of at least 99.9%.

BRIEF DESCRIPTION OF THE DRAWINGS, THE SEQUENCE LISTING, AND THEBIOLOGICAL DEPOSITS

The invention can be more fully understood from the figures, thesequence listing, the biological deposits, and the detailed descriptionthat together form this application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ¹³C NMR spectrum of the resulting glycolonitrile solutionfrom Comparative Example A, qualitatively indicating the purity of theglycolonitrile product. The ¹³C NMR spectrum shows the majorglycolonitrile ¹³C resonances at about δ 48 and 119 ppm. There are alsosubstantial resonances around δ 80-90 ppm for unreacted formaldehyde andaround δ60 ppm for other by-product species derived from unreactedformaldehyde.

FIG. 2 shows ¹³C NMR spectrum of the resulting glycolonitrile solutionfrom Example 1, qualitatively indicating the purity of theglycolonitrile product. The major resonances for glycolonitrile at aboutδ 48 and 119 ppm are observed. The resonances around δ 80-90 ppm evidentin FIG. 1 for unreacted formaldehyde are noticeably reduced in FIG. 2.However, the resonances around δ 60 ppm for by-products derived fromunreacted formaldehyde remain, most likely due to the initialformaldehyde reactor charge.

FIG. 3 shows ¹³C NMR spectrum of the resulting glycolonitrile solutionfrom Example 2, qualitatively indicating the purity of theglycolonitrile product. The major resonances for glycolonitrile at aboutδ 48 and 119 ppm are evident in FIG. 3, while the levels of impuritiesare substantially reduced from the levels observed in FIG. 1 and FIG. 2.

FIG. 4 shows ¹³C NMR spectrum of the resulting glycolonitrile solutionfrom Example 3, qualitatively indicating the purity of theglycolonitrile product. The major resonances for glycolonitrile at aboutδ 48 and 119 ppm are evident in FIG. 4, while the levels of impuritiesare substantially reduced from the levels observed in FIG. 1 and FIG. 2.FIG. 4 also clearly shows the resonance at δ 49 ppm for the methanolfrom the formalin feed used in Example 3.

FIG. 5 shows the ¹³C NMR spectrum of the composite sample produced bymixing the 5 concentrated glycolonitrile samples prepared in Examples4-8, quantitatively indicating the purity of the glycolonitrile product.The quantitative ¹³C NMR analysis was performed on the composite sampleto determine the purity of the glycolonitrile produced.

FIG. 6 shows ¹³C NMR spectrum of the resulting glycolonitrile solution,qualitatively indicating the purity of the glycolonitrile productproduced in Example 9.

FIG. 7 shows ¹³C NMR spectrum of the resulting glycolonitrile solution,qualitatively indicating the purity of the glycolonitrile productproduced in Example 10.

SEQUENCE LISTING

The following sequence descriptions and sequences listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219 (No. 2):345-373 (1984) which are herein incorporated byreference. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the nucleotide sequence of a primer 165 used to amplifythe Acidovorax facilis 72W nitrilase coding sequence. The amplified PCRproduct was subsequently cloned into pUC19 (New England Biolabs,Beverly, Mass.; GenBank® L09137) to create plasmid pSW138.

SEQ ID NO: 2: is the nucleotide sequence of a primer 166 used to amplifythe Acidovorax facilis 72W nitrilase coding sequence. The amplified PCRproduct was subsequently cloned into pUC19 (New England Biolabs,Beverly, Mass.; GenBank® L09137) to create plasmid pSW138.

SEQ ID NO: 3 is the nucleotide sequence of a primer used to amplify theAcidovorax facilis 72W nitrilase.

SEQ ID NO: 4 is the nucleotide sequence of a primer used to amplify theAcidovorax facilis 72W nitrilase.

SEQ ID NO: 5 is the nucleotide sequence of the Acidovorax facilis 72Wnitrilase coding sequence comprising a change in the start codon fromTTG to ATG to facilitate recombinant expression in E. coli.

SEQ ID NO: 6 is the deduced amino acid sequence of the Acidovoraxfacilis 72W nitrilase encoded by the nucleotide sequence of SEQ ID NO: 5comprising a change in the start codon from TTG to ATG to facilitaterecombinant expression in E. coli.

SEQ ID NO: 7 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201Q; Leu→Gln).

SEQ ID NO: 8 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 7) comprising a single amino acid substitution at residueposition 201 (Leu201→Gln) of the A. facilis 72W nitrilase.

SEQ ID NO: 9 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201A; Leu→Ala).

SEQ ID NO: 10 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 9) comprising a single amino acid substitution at residueposition 201 (Leu201→Ala) of the A. facilis 72W nitrilase.

SEQ ID NO: 11 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201C; Leu→Cys).

SEQ ID NO: 12 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 11) comprising a single amino acid substitution at residueposition 201 (Leu201→Cys) of the A. facilis 72W nitrilase.

SEQ ID NO: 13 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201T; Leu→Thr).

SEQ ID NO: 14 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 13) comprising a single amino acid substitution at residueposition 201 (Leu201→Thr) of the A. facilis 72W nitrilase.

SEQ ID NO: 15 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201G; Leu→Gly).

SEQ ID NO: 16 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 15) comprising a single amino acid substitution at residueposition 201 (Leu201→Gly) of the A. facilis 72W nitrilase.

SEQ ID NO: 17 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201H; Leu→His).

SEQ ID NO: 18 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 17) comprising a single amino acid substitution at residueposition 201 (Leu201→His) of the A. facilis 72W nitrilase.

SEQ ID NO: 19 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201K; Leu→Lys).

SEQ ID NO: 20 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 19) comprising a single amino acid substitution at residueposition 201 (Leu201→Lys) of the A. facilis 72W nitrilase.

SEQ ID NO: 21 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201N; Leu→Asn).

SEQ ID NO: 22 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 21) comprising a single amino acid substitution at residueposition 201 (Leu201→Asn) of the A. facilis 72W nitrilase.

SEQ ID NO: 23 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 201 (L201S; Leu→Ser).

SEQ ID NO: 24 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 23) comprising a single amino acid substitution at residueposition 201 (Leu201→Ser) of the A. facilis 72W nitrilase.

SEQ ID NO: 25 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168K; Phe→Lys).

SEQ ID NO: 26 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 25) comprising a single amino acid substitution at residueposition 168 (Phe168→Lys) of the A. facilis 72W nitrilase.

SEQ ID NO: 27 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168M; Phe→Met).

SEQ ID NO: 28 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 27) comprising a single amino acid substitution at residueposition 168 (Phe168→Met) of the A. facilis 72W nitrilase.

SEQ ID NO: 29 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168T; Phe→Thr).

SEQ ID NO: 30 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 29) comprising a single amino acid substitution at residueposition 168 (Phe168→Thr) of the A. facilis 72W nitrilase.

SEQ ID NO: 31 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 168 (F168V; Phe→Val).

SEQ ID NO: 32 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 31) comprising a single amino acid substitution at residueposition 168 (Phe168→Val) of the A. facilis 72W nitrilase.

SEQ ID NO: 33 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 210 (T210A; Thr→Ala).

SEQ ID NO: 34 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 33) comprising a single amino acid substitution at residueposition 210 (Thr210→Ala) of the A. facilis 72W nitrilase.

SEQ ID NO: 35 is the nucleotide sequence of an A. facilis 72W nitrilasemutant comprising a codon change which resulted in a single amino acidsubstitution at residue position 210 (T210C; Thr→Cys).

SEQ ID NO: 36 is the deduced amino acid sequence of the mutant nitrilase(SEQ ID NO: 35) comprising a single amino acid substitution at residueposition 210 (Thr210→Cys) of the A. facilis 72W nitrilase.

SEQ ID NO: 37 is the nucleotide sequence of an A. facilis 72W nitrilasegene expressed in E. coli SS1001 (ATCC PTA-1177; U.S. Pat. No. 6,870,038herein incorporated by reference).

SEQ ID NO: 38 is the deduced amino acid sequence of the A. facilis 72Wnitrilase (SEQ ID NO: 33) expressed in E. coli SS1001 (ATCC PTA-1177).

SEQ ID NO: 39 is the amino acid sequence of catalytic region conservedamong polypeptides having nitrilase activity.

Biological Deposits

The following biological deposits have been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure:

Depositor Identification Int'l. Depository Reference Designation Date ofDeposit Acidovorax facilis 72W ATCC 55746 8 Mar. 1996 E. coli SS1001ATCC PTA-1177 11 Jan. 2000

As used herein, “ATCC” refers to the American Type Culture CollectionInternational Depository Authority located at ATCC, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. The “International DepositoryDesignation” is the accession number to the culture on deposit withATCC.

The listed deposits will be maintained in the indicated internationaldepository for at least thirty (30) years and will be made available tothe public upon the grant of a patent disclosing it. The availability ofa deposit does not constitute a license to practice the subjectinvention in derogation of patent rights granted by government action.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

An integrated process for producing glycolic acid from formaldehyde andhydrogen cyanide is provided. The process begins with the synthesis ofglycolonitrile by contacting heat-treated formaldehyde and hydrogencyanide at a temperature suitable for glycolonitrile synthesis. Theproduct of this reaction is an aqueous solution of glycolonitrile havingfewer impurities (e.g., unreacted monomeric “free” formaldehyde). Thehigh-purity glycolonitrile is subsequently contacted with an enzymecatalyst having nitrilase activity whereby an aqueous solutioncomprising ammonium glycolate is produced. High purity glycolic acid(having a purity of at least 99.9%) can then be obtained from theammonium glycolate using a method such as ion exchange.

In one embodiment, the process uses an enzyme catalyst comprising apolypeptide providing a significant (i.e., at least 1.5-fold)improvement nitrilase activity when compared to the nitrilase activityof the Acidovorax facilis 72W (ATCC 55746) nitrilase (SEQ ID NO: 6)under identical reaction conditions. The improvement in catalystactivity increases catalyst productivity and volumetric productivity,decreasing the overall cost of manufacturing high purity glycolic acid.

In another embodiment, the enzyme catalyst having a significantimprovement in nitrilase activity comprises a polypeptide having anamino acid sequence of SEQ ID NO: 6 with at least one amino acidsubstitution selected from the group consisting of:

-   -   (a) a substitution at amino acid residue 168 with lysine,        methionine, threonine or valine; and    -   (b) a substitution at amino acid residue 201 with glutamine,        glycine, histidine, lysine, asparagine, serine, alanine,        cysteine, or threonine.

In a further embodiment, the enzyme catalyst provides a catalystproductivity of at least 300 grams of glycolic acid per gram dry cellweight of enzyme catalyst.

The aqueous solution comprising glycolonitrile is converted into theammonium salt of the glycolic acid (ammonium glycolate) using an enzymecatalyst having nitrilase activity where the pH of the reaction isgenerally maintained between about pH 6 and about pH 8 (pKa of glycolicacid ˜3.83). A variety of methods can than be used to obtain glycolicacid from the aqueous solution of ammonium glycolate including, but notlimited to ion exchange, electrodialysis, reactive solvent extraction,thermal decomposition (salt cracking), alcoholysis, and combinationsthereof. One of skill in the art will recognize that the preferredisolation/purification strategy for glycolic acid is determined bylocation-specific factors such as energy cost, by-product values, wastedisposal costs, capital equipment investment, financing opportunities,as well as local, state and national laws, rules and regulationsincluding environmental considerations.

Several process conditions are also provided that improve enzymecatalyst stability and productivity, thereby decreasing catalyst costand overall cost of manufacture. These process conditions include 1) theuse of additives to stabilize enzyme catalyst activity, 2) running theenzymatic catalyzed reaction under substantially oxygen free conditions,and 3) controlling the feed rate of glycolonitrile to the reactionmixture so that a targeted concentration of glycolonitrile ismaintained.

DEFINITIONS

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

As used herein, the term “recovering” means isolating, purifying, ortransferring the product formed by the present process. Methods toisolate and purify the product(s) from the reaction mixture are wellknown in the art may include, but are not limited to selectiveprecipitation, crystallization, filtration, reactive solvent extraction,ion exchange, electrodialysis, polymerization, distillation, thermaldecomposition, alcoholysis, column chromatography, and combinationsthereof. In one embodiment, the term “recovering” may also includetransferring the product mixture (typically after filtering out theenzyme catalyst) to another reaction to create one or more additionalproducts. In a preferred embodiment, ion exchange is used to recover theglycolic acid.

As used herein, the term “glycolonitrile” is abbreviated as “GLN” and issynonymous with hydroxyacetonitrile, 2-hydroxyacetonitrile,hydroxymethylnitrile, and all other synonyms of CAS Registry Number107-16-4.

As used herein, the term “glycolic acid” is abbreviated as “GLA” and issynonymous with hydroxyacetic acid, hydroxyethanoic acid, and all othersynonyms of CAS Registry Number 79-14-1. The glycolic acid produced bythe present processes may in the form of the protonated carboxylic acidand/or the corresponding ammonium salt.

As used herein, the term “ammonium glycolate” is abbreviated “NH₄GLA”.

As used herein, the term “glycolamide” is the amide derived from thereaction of ammonia with glycolic acid and refers to all other synonymsof compounds having CAS Registry Number 598-42-5.

As used herein, the term “glycolide” refers to the compound of CASRegistry Number 502-97-6.

As used herein, the term “formaldehyde” is abbreviated as “FA” and issynonymous with formic aldehyde, methyl aldehyde, oxomethane, and allother synonyms of CAS Registry Number 50-00-0. Commercially availableformaldehyde is typically comprised of a mixture of monomericformaldehyde (“free formaldehyde”) and various oligomers of formaldehydealong with some methanol (typically about 1 wt % to about 15 wt %).

As used herein, the term “hydrogen cyanide” is synonymous with prussicacid, hydrocyanic acid, and all other synonyms of CAS Registry Number200-821-6.

As used herein, the terms “formaldehyde heat treatment”, “heat-treatedformaldehyde”, “heating the formaldehyde feed stream”, “pre-heatedformaldehyde”, and “an aqueous formaldehyde feed stream that is heated”are used to describe the process of subjecting an aqueous formaldehydesolution to a prescribed temperature for a determinable period of timeprior to reacting with hydrogen cyanide. As used herein, the term“determinable period of time” is used to describe the amount of time theformaldehyde feed stream is heated to the specified temperature. Theoptimal length of time the formaldehyde is heat-treated can be easilydetermined and may be adjusted depending upon the selected temperaturein combination with the specific design of the heat treatment system andthe reactor. The length of the heat treatment is chosen to maximize theamount of monomeric formaldehyde in the heated feed stream. Themonomeric formaldehyde reacts with the hydrogen cyanide to produce aglycolonitrile solution with substantially fewer impurities (i.e.unreacted formaldehyde and impurities associated with polymeric forms offormaldehyde). Typically, the heat treatment period can last from about10 seconds to about 24 hours, preferably about 10 seconds to about 6hours, more preferably about 10 seconds to about 20 minutes, and mostpreferably about 2 minutes to about 10 minutes. In one embodiment, theheat treatment time is about 2 minutes to about 10 minutes in thepresence of a base catalyst. The heated formaldehyde is then promptlyfed to the reaction chamber. The heated formaldehyde is promptly fed tothe reactor and contacted with hydrogen cyanide whereby glycolonitrileat “a temperature suitable for glycolonitrile synthesis”.

As used herein, the term “a temperature suitable for glycolonitrilesynthesis” is used to describe a reaction temperature range suitable forreacting hydrogen cyanide and the heat-treated formaldehyde. In oneembodiment, the reaction temperature is typically about 70° C. or lessin order to minimize glycolonitrile decomposition. In anotherembodiment, the reaction temperature is between about −20° C. to about70° C., preferably about 0° C. to about 70° C., more preferably about 0°C. to about 55° C., even more preferably about 10° C. to about 30° C.,and most preferably about 20° C. to about 25° C.

As used herein, the terms “promptly fed to the reactor” and “promptlyadding the heated formaldehyde” are used to described the time periodbetween the end of the heat treatment period and the initiation of thereaction with hydrogen cyanide, typically less than about 24 hours,preferably less than about 1 hour, more preferably less than about 15minutes, most preferably less than about 5 minutes.

As used herein, the terms “Acidovorax facilis” and “A. facilis” are usedinterchangeably and refer to Acidovorax facilis 72W deposited to theAmerican Type Culture Collection (an international depository authority)having accession number 55746 (“ATCC 55746”). The present mutantnitrilases having improved nitrilase activity were engineered bysubjecting the Acidovorax facilis 72W nitrilase to error-prone PCRand/or targeted saturation mutagenesis.

As used herein, the terms “Escherichia coli” and “E. coli” are usedinterchangeably. Several strains of E. coli suitable for recombinantexpression are described herein including, but not limited to E. coliMG1655 having international depository number ATCC 47076, E. coli FM5having international depository number ATCC 53911, E. coli W3110 havinginternational depository number ATCC 27325, E. coli MC4100 havinginternational depository number ATCC 35695, and E. coli W1485 havinginternational depository number ATCC 12435. In one embodiment, suitableEscherichia coli strains include E. coli FM5 (ATCC 53911) and E. coliMG1655 (ATCC 47076).

As used herein, the terms “E. coli SS1001” or “SS1001” refer to atransformed E. coli strain expressing the Acidovorax facilis 72Wnitrilase having ATCC Accession No. PTA-1177 (U.S. Pat. No. 6,870,038;herein incorporated in its entirety by reference). The recombinantlyexpressed E. coli SS1001 nitrilase (SEQ ID NO: 38) contains 2 minorsequence changes in comparison to the wild-type 72W nitrilase sequence(SEQ ID NO: 6). The start codon was changed from GTG to ATG tofacilitate recombinant expression and an artifact was introduced duringcloning that resulted in a single amino acid change near the C-terminal(Pro367 [CCA]→Ser [TCA]).

As used herein, the term “stabilizing agent” will be used to describematerials that may be added to the enzymatic catalyst reaction mixturethat may help stabilize catalyst activity. The stabilizing agents mayinclude, but are not limited to thiosulfates (e.g. potassiumthiosulfate, K₂S₂O₃), dithionites (e.g., sodium dithionite, Na₂S₂O₄),and cyanide compounds (e.g. HCN, NaCN, KCN, etc.).

As used herein, the terms “suitable aqueous glycolonitrile reactionmixture” and “suitable aqueous reaction mixture” refer to the materialsand water in which the glycolonitrile and enzyme catalyst come intocontact. The components of the suitable aqueous reaction mixture areprovided herein and those skilled in the art appreciate the range ofcomponent variations suitable for this process.

As used herein, the terms “aqueous ammonium glycolate solution”,“aqueous solution comprising ammonium glycolate”, and “aqueous solutionof ammonium glycolate” will be used to describe an aqueous solutioncomprising ammonium glycolate produced by the enzymatic hydrolysis ofglycolonitrile under typical enzymatic reaction conditions (i.e., a pHrange of about 6 to about 8). The aqueous solution of ammonium glycolatecomprises ammonium glycolate at a concentration of at least about 0.1weight percent (wt %) to about 99 wt % ammonium glycolate. In anotherembodiment, the aqueous solution of ammonium glycolate is comprised ofat least about 10 wt % to about 75 wt % ammonium glycolate. In a furtherembodiment, the aqueous solution of ammonium glycolate is comprised ofat least about 20 wt % to about 50 wt % ammonium glycolate. The pH ofthe aqueous solution of ammonium glycolate can be about 2 to about 12,preferably 5 to about 10, more preferably 6 to about 8. The pH may beadjusted as needed prior to initiating process steps related torecovering glycolic acid (in the form of the acid or salt) from theaqueous ammonium glycolate solution.

As used herein, the term “aqueous ammonium glycolate feed stream” willbe used to describe an aqueous solution comprising ammonium glycolatewhen used as a feed stream in a process step used to obtain and/orrecover glycolic acid from the aqueous solution of ammonium glycolate.In one embodiment, the aqueous ammonium glycolate feed stream may alsobe concentrated and/or acidified (typically using a mineral acid such asH₂SO₄) prior to isolation of the final glycolic acid product in thepresent invention. A variety of processing techniques known and the artand several described herein can be used to obtain and/or recoverglycolic acid from the aqueous solution of ammonium glycolate producedby the present method.

As used herein, the term “enzyme catalyst” or “nitrilase catalyst”refers to a catalyst that is characterized by a nitrilase activity (EC3.5.5.7). The enzyme catalyst comprises a polypeptide (“nitrilase”)having nitrilase activity for converting glycolonitrile to glycolic acidand ammonia. A nitrilase enzyme directly converts an aliphatic nitrileto the corresponding carboxylic acid, without forming the correspondingamide as intermediate (see Equation 1). Nitrilases share severalconserved signature domains known in the art including a signaturedomain herein referred to as the “catalytic domain” or “catalyticregion”. This region comprises an essential cysteine residue (e.g.,Cys164 of SEQ ID NO: 6). As such, polypeptides having nitrilase activitycan be identified by the existence of the catalytic domain amino acidsequence (G-Xaa-L-Xaa-C-Xaa-E-Xaa-Xaa-Xaa-Xaa-L; SEQ ID NO: 39 whereinXaa is a non-conserved amino acid).

The enzyme catalyst may be in the form of a whole microbial cell, apermeabilized microbial cell, one or more cell components of a microbialcell extract, partially purified enzyme, or purified enzyme. As usedherein, “recycled enzyme catalyst” refers to an enzyme catalyst that isreused as an enzyme catalyst in batch reactions.

As used herein, the terms “improved nitrilase”, “mutant nitrilase”,“Acidovorax facilis 72W mutant nitrilase”, and “protein engineerednitrilase” will be used interchangeably to refer to the present enzymecatalyst comprising a polypeptide providing a significant improvement innitrilase activity towards the conversion of glycolonitrile to glycolicacid in comparison to the activity of the A. facilis 72W nitrilase (SEQID NO: 6) under identical reaction conditions. In one embodiment, theimprovement in nitrilase activity can be determined by comparing thenitrilase activity of present nitrilases against the nitrilase activityof the A. facilis 72W nitrilase when recombinantly expressed (usingidentical expression systems) and assayed under essentially identicalreaction conditions. SDS-PAGE analysis indicated that protein expressionlevels between the present mutants and their respective controls (SEQ IDNO: 6) were essentially identical. As such, improvements in nitrilaseactivity are attributed to structural modifications to the native A.facilis 72W nitrilase.

As used herein, the terms “catalyst productivity” and “enzyme catalystproductivity” refer to the total amount of product produced per gram ofcatalyst. In the present invention, the catalyst is a nitrilase enzyme(EC 3.5.5.7) and the product formed is glycolic acid and/or ammoniumglycolate (depending upon the pH of the reaction). In general, thepresent methods are conducted under essentially pH neutral conditions sothat the glycolic acid produced is predominantly in the form of thecorresponding salt of glycolic acid (i.e. ammonium glycolate).Generally, in batch reactions with catalyst recycle, the catalystactivity decreases with each recycle reaction (enzyme inactivation).

The term “nitrilase activity” refers to the enzyme activity per unitmass (for example, milligram) of protein, dry cell weight, or beadweight (immobilized catalyst) when converting glycolonitrile to glycolicacid (or the corresponding ammonium glycolate). Comparisons in nitrilaseactivity were measured proportional to the dry cell weight or beadweight. Since nitrilase expression levels between the A. facilis 72Wcontrols (transformed microbial cells expressing the A. facilis 72Wnitrilase having the amino acid sequence of SEQ ID NO: 6) and theimproved mutants (transformed microbial cells expressing the presentnitrilases) were indistinguishable as quantified using laserdensitometry analysis of an SDS-PAGE gel, comparisons and reportedimprovements in nitrilase activity were measured relative to dry cellweight (dcw) or bead weight (bw).

As used herein, the term “one unit of enzyme activity” or “one unit ofnitrilase activity” or “U” is defined as the amount of enzyme activityrequired for the production of 1 μmol of glycolic acid product perminute (GLA U/g dry cell weight or bead weight) at a specifiedtemperature (e.g. 25° C.).

As used herein, the terms “relative nitrilase activity”, “improvednitrilase activity”, and “relative improvement in nitrilase activity”refers to the nitrilase activity expressed as a multiple (or fraction)of a reference (control) nitrilase activity. The present mutantnitrilases exhibit a significant improvement in nitrilase activityrelative to the nitrilase activity observed with native Acidovoraxfacilis 72W nitrilase. In the present invention, a “significantimprovement” in relative nitrilase activity is an improvement of atleast 1.5-fold higher nitrilase activity in comparison to the nitrilaseactivity of the control (A. facilis 72W nitrilase; SEQ ID NO: 6) underidentical reaction conditions. In another embodiment, the improvement isat least 2-fold higher nitrilase activity in comparison to the nitrilaseactivity of the control under identical reaction conditions. In afurther embodiment, the improvement is at least 4-fold higher nitrilaseactivity in comparison to the nitrilase activity of the control underidentical reaction conditions.

As used herein, the term “initial reaction rate” is a measurement of therate of conversion of glycolonitrile to glycolic acid under the statedreaction conditions, where the measurement of reaction rate begins uponthe initial addition of glycolonitrile to the reaction mixture, andwhere the reaction rate is measured over a period of time where theconcentration of glycolonitrile remains above ca. 50 millimolar (mM)during the course of the reaction. The reaction rate is measured as thechange in concentration of glycolic acid produced per unit time (e.g.,mole glycolic acid/L/min or mM glycolic acid/hour).

As used herein, the terms “recombinant organism”, “transformed host”,“transformant”, “transgenic organism”, and “transformed microbial host”refer to a host organism having been transformed with heterologous orforeign DNA. The recombinant organisms of the present invention expressforeign coding sequences or genes that encode active nitrilase enzyme.“Transformation” refers to the transfer of a DNA fragment into the hostorganism. The transferred DNA fragment can be chromosomally orextrachromosomally incorporated (i.e., via a vector) into the hostorganism. As used herein, the term “transformation cassette” refers to aspecific fragment of DNA containing a set of genetic elementsconveniently arranged for insertion into a host cell, usually as part ofa plasmid. As used herein, the term “expression cassette” refers to aspecific fragment of DNA containing a set of genetic elementsconveniently arranged for insertion into a host cell, usually as part ofa plasmid, that also allows for enhanced gene expression in the host.

As used herein, the terms “nucleic acid fragment” and “nucleic acidmolecule” refer to DNA molecule that may encode an entire gene, codingsequence, and/or regulatory sequences preceding (5′, upstream) orfollowing (3′, downstream) the coding sequence. In one aspect, thepresent nucleic acid molecules encode for polypeptides having nitrilaseactivity.

As used herein, the term “gene” refers to a nucleic acid molecule thatexpresses a specific protein. As used herein, it may or may notincluding regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Chimeric gene”refers to any gene that is not a native gene, comprising regulatory andcoding sequences that are not found together in nature. Accordingly, achimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

As used herein, the term “coding sequence” refers to a DNA sequence thatcodes for a specific amino acid sequence. As used herein, “suitableregulatory sequences” refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. Promoters that cause a gene to beexpressed in most cell types at most times or under most environmentalconditions are commonly referred to as “constitutive promoters”.Promoters that cause a gene to be expressed only in the presence of aparticular compound or environmental condition are commonly referred toas “inducible promoters”. Since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid molecule so that thefunction of one sequence is affected by the other. For example, apromoter is operably linked with a coding sequence when it is capable ofaffecting the expression of that coding sequence (i.e., that the codingsequence is under the transcriptional control of the promoter). Codingsequences can be operably linked to regulatory sequences in sense orantisense orientation.

As used herein, the term “3′ non-coding sequences” refers to DNAsequences located downstream of a coding sequence and includepolyadenylation recognition sequences (normally limited to eukaryotes)and other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal (normallylimited to eukaryotes) is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor.

The skilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in using nucleotide codons to specify a given aminoacid. Therefore, when synthesizing a gene for improved expression in ahost cell, it is desirable to design the gene such that its codon usagereflects the preferred codon bias of the host cell. A survey of genesderived from the host cell where sequence information is available candetermine its codon bias. Codon-optimization is well known in the artand has been described for various systems including, but not limited toyeast (Outchkourov et al., Protein Expr Purif, 24(1):18-24 (2002)) andE. coli (Feng et al., Biochemistry, 39(50):15399-15409 (2000)).

Definitions Applying to Reactive Solvent Extraction

As used herein, the term “reactive extraction process” refers to theprocess of contacting (i.e., mixing) an aqueous solution comprisingglycolic acid (i.e., second phase) with a water-immiscible organicsolvent (i.e., first phase) whereby the glycolic acid reacts with thetertiary trialkylamine to form an glycolic acid:trialkylamine complex.The complex is soluble in the organic phase, thereby extracting glycolicacid from the aqueous phase (i.e., second phase comprising substantialamounts of impurities) into the organic phase, forming a “glycolicacid-loaded first phase”. The glycolic acid-loaded first phase issubsequently isolated from the aqueous second phase. A back extractionprocess is then used to extract the glycolic acid from the organic phaseback into an aqueous phase (i.e. the “third phase”). The length of timeand temperature used for the reactive extraction process may be adjustedto optimize the extraction efficiency. In one embodiment, the mixingperiod of the first and second phase is about 5 minutes to about 8hours, preferably about 5 minutes to about 1 hour, more preferably about10 minutes to about 30 minutes. The temperature may range from about 5°C. to about 90° C., more preferably about 25° C. to about 75° C., andmost preferably about 25° C. to about 50° C.

As used herein, the term “back extraction process” refers to the processof contacting a water-immiscible organic solvent comprising glycolicacid (i.e. “glycolic acid-loaded first phase”) with water (i.e. “thirdphase”) to extract the glycolic acid from the organic phase into theaqueous phase. In one embodiment, the third phase is deionized water.After back extraction, the third phase comprises a substantiallypurified form of glycolic acid (substantially less mineral salts andother impurities). The glycolic acid in the third phase can beoptionally isolated using a variety of techniques know in the art. Thelength of time and temperature used for the back extraction process maybe adjusted to optimize the extraction efficiency. In one embodiment,the mixing period of the “glycolic acid-loaded first phase” and aqueousphase (i.e., third phase) is about 10 minutes to about 8 hours,preferably about 30 minutes to about 4 hours, more preferably about 30minutes to about 60 minutes. Typically, the back extraction processoccurs under pressurized conditions under a non-reactive gas (i.e.,nitrogen) blanket. The pressure in the back extraction chamber may bevaried but is typically less than about 100 psi (less than about 690kPa). The temperature may range from about 5° C. to about 150° C.,preferably about 100° C. to about 150° C., and most preferably about125° C. to about 140° C.

As used herein, the terms “water-immiscible organic solvent” and “firstphase” are used to describe an organic solvent mixture comprising atleast one tertiary trialkylamine having the formula:

wherein R₁, R₂, and R₃ are independently C8 to C12 alkyl group; and

at least one diluent selected from the group consisting of methylisobutyl ketone, 1-octanol, 1-decanol, methylene chloride,1-chlorobutane, chlorobenzene, chloroform, kerosene, toluene, mixedxylenes, tributyl phosphate, and mixtures thereof.

As used herein, the term “second phase” refers to an aqueous solutioncomprising glycolic acid having a pH of about 4 or less, preferablyabout 3 or less, and most preferably about 1 to about 2. The “secondphase” is prepared by adjusting the pH of an aqueous solution comprisingammonium glycolate with a strong mineral acid, such as H₂SO₄. However,the addition of the strong acid increases the amount of mineral salts(an undesirable impurity) in the second phase. Extraction (i.e.,reactive solvent extraction) of the glycolic acid from the second phaseinto an organic phase (typically as a complex with a trialkyl amine)separates the glycolic acid from the mineral salt impurities.

Definitions Applying to Thermal Salt Cracking

As used herein, the terms “direct deammoniation”, “thermal saltcracking”, “thermal salt decomposition”, “thermal decomposition”, and“salt cracking” refer to the process where a heat treatment is appliedto the ammonium salt of the organic acid (i.e., ammonium glycolate) fora period of time to decompose the ammonium salt of the acid into thefree organic acid and ammonia. In the present invention, thermaldecomposition is used to produce primarily glycolic acid and ammonia.Various byproducts, such as glycolamide and oligomers of glycolic acid,may also be formed in the product mixture.

As used herein, the term “substantially anhydrous salt of ammoniumglycolate” will refer to the resulting salt of ammonium glycolate (aliquid at room temperature) formed after removing at least about 90 wt%, preferably at least about 95 wt %, more preferably at least about99%, and most preferably at least about 99.5 wt % of the free water fromthe aqueous ammonium glycolate feed stream. Typically, the“substantially anhydrous salt of ammonium glycolate” is formed an theinitial step when using direct deammoniation (“thermal salt cracking”;see copending U.S. application 60/638,148) to obtain glycolic acid froman aqueous solution comprising ammonium glycolate.

As used herein, the term “free water” will refer to the water that isreadily removed from the feed stream prior to thermal salt cracking,where some small but measurable amount of water may not be removed fromthe feed stream prior to thermal salt cracking (for example, water ofhydration of the ammonium glycolate salt). As used herein, the term“molten salt of ammonium glycolate” will refer to the substantiallyanhydrous salt of ammonium glycolate that is thermally decomposed in thepresence of a strong vacuum.

As used herein the term “first liquid product mixture” or “first liquidproduct mixture comprising glycolic acid” refers to the product obtainedafter thermally decomposing a substantially anhydrous salt of ammoniaglycolate as described in the present methods. The first liquid productmixture comprises glycolic acid, oligomers of glycolic acid,glycolamide, ammonium salts of oligomers of glycolic acid, and unreactedammonium glycolate.

As used herein, the term “second liquid product mixture” refers to theproduct obtained by the process of 1) adding water to said first productmixture to form a rehydrated first product mixture; and 2) heating saidrehydrated first product mixture whereby a portion of the glycolic acidoligomers are hydrolyzed into free glycolic acid. Subsequent processingto obtain glycolic acid from the partially deammoniated products (i.e.,first product mixture or second product mixture) produces significantlyless waste in comparison to ammonium glycolate that was not thermallydecomposed prior to processing.

As used herein, the term “rehydrated first liquid product mixture”refers to the aqueous product obtained when water is added to the firstliquid product mixture produced by the present methods.

Definitions Applying to Alcoholysis

As used herein, the term “alcoholysis” refers to the process of reactingan aqueous solution of a ammonium glycolate with a heated alcohol vaporthat acts as both an esterifying agent and stripping gas, producing avapor product stream comprising the glycolic acid ester (see copendingU.S. provisional patent application 60/638,126). As used herein, theterm “methanolysis” refers to the process of alcoholysis wherein thealcohol is methanol, and the corresponding ester is methyl glycolate.

As used herein, the terms “heated alcohol vapor” and “alcohol vapor feedstream” refer to the heated alcohol vapor that is contacted with theaqueous solution comprising ammonium glycolate whereby glycolic acidester is produced; wherein the carboxylic acid ester product is in thevapor product phase (“alcoholysis”). In one embodiment, the heatedalcohol vapor is a heated methanol vapor and the resulting ester vaporis a methyl glycolate vapor.

As used herein, the terms “first vapor product stream”, “vapor productstream”, and “alcohol vapor product stream” refer to the vapor productstream comprising the heated alcohol vapor and the glycolic acid ester(vapor) produced by alcoholysis. Methods to recover/isolate glycolicacid ester (e.g., methyl glycolate) from the first vapor product streamare well known in the art and include, but are not limited to membraneseparation, adsorption, direct or indirect contact condensation (e.g.,partial condenser), use of distillation column(s), and combinations. Therecovered glycolic acid ester (liquid) is collected in the “first liquidproduct stream”. As used herein, the term “first liquid product stream”refers to the liquid product comprising the glycolic acid esterrecovered from the first vapor product stream (produced during theprocess of alcoholysis). In one embodiment, a partial condenser is usedto recover the glycolic acid ester from the first vapor product streamwhere the most of the heated alcohol vapor passes through the partialcondenser (“hot condenser”) and is subsequently recovered using a totalcondenser (“cold condenser”). The recovered alcohol may be recycled andreused at the starting material for the heated vapor feed stream. Anyammonia or water recovered may be optionally removed from the recoveredalcohol prior to being recycled.

Synthesis of Glycolonitrile from Formaldehyde and Hydrogen Cyanide

Methods to synthesize glycolonitrile by reacting aqueous solutions offormaldehyde and hydrogen cyanide have previously been reported (U.S.Pat. No. 2,175,805; U.S. Pat. No. 2,890,238; and U.S. Pat. No.5,187,301; Equation 3).

Concentrated aqueous solutions of formaldehyde (e.g., 37 wt % solutionscommercially known as formalin) typically are comprised of freeformaldehyde and various oligomers of formaldehyde (for example,paraformaldehyde and trixoxymethylene). The presence of formaldehydeoligomers can influence overall conversion to glycolonitrile. Hence, amethod to pre-treat the formaldehyde that transforms formaldehydeoligomers to more free formaldehyde in the feed stream prior to reactingwith hydrogen cyanide should increase the yield of glycolonitrile andshould decrease the conversion of unwanted secondary products producedfrom the oligomers.

Jacobson (U.S. Pat. No. 2,175,805) discloses a method of obtaining pureglycolonitrile by the reaction of hydrogen cyanide and formaldehyde inthe presence of an acidic compound followed by distillation atsubatmospheric pressure (vacuum distillation step conducted at about125° C.). The reactants are preferably mixed “in the cold” (i.e., below26° C. to maintain the hydrogen cyanide in liquid form). Also describedin U.S. Pat. No. 2,175,805 is the observation that 1) glycolonitriledecomposes at ambient temperature, and 2) glycolonitrile contacted withbases decomposes violently within hours at ambient temperature. Jacobsondoes not disclose pre-treatment of the concentrated aqueous formaldehydefeed prior to reacting with hydrogen cyanide.

Sexton (U.S. Pat. No. 2,890,238) discloses a method of preparingglycolonitrile in which formaldehyde is fed into an aqueous solution ofHCN. The reaction is run “with efficient reflux or a closed pressuresystem, with the reaction allowed to go as high as 100° C.” However, asdescribed in Jacobson, glycolonitrile decomposes at room temperature. Areaction run at temperatures as high as 100° C. would be expected toresult in an increase in the decomposition of the glycolonitrile.Similar to Jacobson, Sexton does not describe a method to pre-treat theformaldehyde prior to reacting with hydrogen cyanide.

Cullen et al. (U.S. Pat. No. 5,187,301) discloses a method for makingiminodiacetonitrile from glycolonitrile. This reference describes howglycolonitrile can be formed in a process (either batch or continuous)by maintaining the pH of the formaldehyde above about 3, preferably inthe range of about 5-7, most preferably about 5.5, with suitable acidsand bases. The formaldehyde is then reacted with hydrogen cyanide in atemperature range of about 20 to 80° C., preferably about 30° C., toform glycolonitrile. However, as shown in the present examples, areaction run within the conditions specified in Cullen et al. results ina significant amount of unreacted free formaldehyde after 2 hours ofreaction time.

All of the above mentioned methods produce a purity of glycolonitrilethat typically requires extensive processing steps, such as distillativepurification, to remove some of the secondary products (impurities).Many of the impurities found in glycolonitrile, such as unreactedformaldehyde, have been reported to interfere with the enzymaticconversion to glycolic acid by inactivating the enzyme catalyst (U.S.Pat. No. 5,756,306; U.S. Pat. No. 5,508,181; and U.S. Pat. No.6,037,155; herein incorporated in their entirety by reference).

Concentrated aqueous formaldehyde solutions are typically comprised ofmonomeric formaldehyde (“free formaldehyde”, the desired substrate forthe reaction) and oligomers of formaldehyde. Applying a heat treatmentto the formaldehyde feed stream prior to reacting with hydrogen cyanideimproves the purity of the resulting glycolonitrile product (See“Comparative Example A” and Examples 1-10 of the present application andcopending U.S. Provisional Application 60/638,127; herein incorporatedby reference). The reaction of formaldehyde with hydrogen cyanide istemperature-controlled to minimize glycolonitrile decomposition. Thereaction product formed is an aqueous solution of glycolonitrilecomprising significantly less unreacted formaldehyde when compared to areaction product obtained without pre-heating the aqueous formaldehydefeed stream.

The resulting aqueous glycolonitrile solution (see Examples 4-8; Table 1where glycolonitrile purity was greater than 99.9%) requires fewer, ifany, post reaction purification steps (such as distillativepurification), thus reducing the cost of producing glycolonitrile thatis suitable for enzymatic synthesis of glycolic acid. In one embodiment,the glycolonitrile produced in the present process does not require anypost reaction purification steps prior to being contacted with an enzymecatalyst having nitrilase activity. Additionally, reducing the amount ofunreacted formaldehyde in the aqueous glycolonitrile solution used forenzymatic synthesis of glycolic acid should extend the enzymaticcatalyst's lifespan (i.e., the number of recycle reactions). Thisimproves the catalyst's productivity and reduces the cost for preparingglycolic acid. In one embodiment, the invention yields a glycolonitrileproduct that may be used directly for enzymatic conversion withoutpurification, significantly reducing the cost of producing glycolicacid.

Suitable Reaction Conditions to Produce Improved Purity Glycolonitrile

The present method to produce glycolic acid includes a process toproduce aqueous glycolonitrile by contacting heat-treated formaldehydeand hydrogen cyanide at reaction temperature suitable for glycolonitrilesynthesis (see Comparative Example A and Examples 1-10). Theformaldehyde is heated prior to reacting with the hydrogen cyanide tomake glycolonitrile. The starting concentration of the formaldehyde istypically an aqueous solution of about 5 wt % to about 70 wt %formaldehyde. In one embodiment, the formaldehyde feed stream iscomprised of about 20 wt % to about 55 wt % formaldehyde. In anotherembodiment, the formaldehyde feed stream is comprised of about 37 wt %formaldehyde (e.g., formalin). The formaldehyde feed stream mayoptionally be comprised of about 0.1 wt % to about 15 wt % (typically6-8 wt %) methanol (an additive typically found in some 37 wt %solutions).

A base catalyst (KOH, NaOH, etc.) may be added to the aqueousformaldehyde solution prior to heating the aqueous formaldehyde feedstream. As exemplified herein, sodium hydroxide may be added to theaqueous formaldehyde feed stream prior to heating the formaldehyde feedstream. In one embodiment, the molar ratio of NaOH:formaldehyde in theheated aqueous formaldehyde feed stream is about 1:50 to about 1:2000.In another embodiment, the molar ratio of NaOH:HCHO in the heatedaqueous formaldehyde feed stream is about 1:100 to about 1:2000.

The formaldehyde feed stream may be heated to a temperature of about 35°C. to about 200° C. In one embodiment, the formaldehyde feed stream isheated to a temperature of about 90° C. to about 150° C. In anotherembodiment, the formaldehyde feed stream is heated to a temperature ofabout 100° C. to about 125° C.

The optimal length of time the formaldehyde is heat-treated can beeasily determined and may be adjusted depending upon the selectedtemperature in combination with the specific design of the heattreatment system and the reactor. The length of the heat treatment ischosen to maximize the amount of monomeric formaldehyde in the heatedfeed stream. Hence, the amount of time the formaldehyde is heated to thedesired temperature may be easily determined. As used herein, the term“determinable period of time” is used to describe the amount of time theformaldehyde feed stream is heated to the specified temperature.Typically, the heat treatment period can last from about 10 seconds toabout 24 hours, preferably about 10 seconds to about 6 hours, morepreferably about 10 seconds to about 20 minutes, and most preferablyabout 2 minutes to about 10 minutes. In one embodiment, the heattreatment time is about 2 minutes to about 10 minutes in the presence ofa base catalyst. The heated formaldehyde is then promptly fed to thereaction chamber.

The hydrogen cyanide feed stream is typically added at a rate sufficientto maintain a slight molar excess of hydrogen cyanide relative to theamount of formaldehyde added to the reaction chamber. In one embodiment,the molar ratio of hydrogen cyanide to formaldehyde is at least about1.01:1, preferably no greater than about 10:1. In another embodiment,the molar ratio of HCN to formaldehyde is about 1.01:1, more preferablyno greater than about 2:1. In a further embodiment, the molar ratio ofHCN to formaldehyde is about 1.05:1 to about 1.15:1.

The reaction chamber may optionally be pre-charged with hydrogen cyanideso that the formaldehyde is immediately in contact with the hydrogencyanide upon addition to the reaction chamber. Pre-charging the reactionchamber with hydrogen cyanide aids in maintaining the slight excess ofhydrogen cyanide during the reaction. One skilled in the art recognizesthat when an HCN pre-charge is used, the molar ratio of the HCN toformaldehyde rapidly transitions from infinity to the more sustainableration of 10:1 or less, preferably 2:1 or less, more preferably about1.01:1 to about 1.15:1, and most preferably about 1.01:1 to about1.05:1.

The temperature of the reaction chamber (i.e., the temperature suitablefor producing glycolonitrile) is typically maintained about 70° C. orless in order to minimize glycolonitrile decomposition. In oneembodiment, the reaction temperature is between about −20° C. to about70° C. In another embodiment, the reaction temperature is about 0° C. toabout 70° C. In yet another embodiment, the reaction chamber temperatureis about 0° C. to about 55° C. In a further embodiment, the reactiontemperature is about 10° C. to about 30° C. In yet a further embodiment,the reaction temperature is about 20° C. to about 25° C.

Atmospheric pressure is satisfactory for carrying out the reaction offormaldehyde and hydrogen cyanide, and hence pressures of from about 0.5to about 10 atmospheres (50.7 kPa to 1013 kPa) are preferred. Higherpressures, up to 20,000 kPa or more may be used, if desired, but anybenefit that may be obtained thereby would probably not justify theincreased cost of such operations.

The pH in the glycolonitrile synthesis reaction chamber is typicallyabout 3 to about 10, preferably about 5 to about 8.

The present glycolonitrile synthesis reaction can be run in continuous,batch, or fed batch mode. The fed batch reaction typically is run forabout 10 seconds to about 24 hours, preferably about 30 minutes to about8 hours, more preferably about 0.5 hours to about 4 hours.

Stabilization of Glycolonitrile Under Acidic Conditions

The glycolonitrile produced by the present process steps is subsequentlycontacted with the present enzyme catalysts and converted into glycolicacid (typically in the form of the ammonium salt of glycolic acid). Inone embodiment, the glycolonitrile produced by the present process stepsmay be stabilized (typically when the glycolonitrile is to be stored fora period of time prior to enzymatic conversion) with a mineral acid(e.g., HCl, H₂SO₄, or H₃PO₄) to maintain the pH of the glycolonitrilebelow 7 (glycolonitrile has been reported to decompose under basicconditions). The need to stabilize the glycolonitrile determines on avariety of factors including storage time and conditions. One of skillin the art can easily determine whether or not the glycolonitrileproduced using the present process steps should be acid stabilized. Inanother preferred embodiment, glycolic acid is added to theglycolonitrile mixture obtained by the present method to maintain the pHof the glycolonitrile below 7. In a further embodiment, the amount ofglycolic acid added is sufficient to maintain the pH of theglycolonitrile below about 6, preferably below about 5, more preferablybelow about 4, and most preferably below about 3.5. Stabilization withglycolic acid is a preferred embodiment in the instance where theglycolonitrile is subsequently converted to glycolic acid using anenzyme catalyst. The use of glycolic acid to adjust the pH in thisinstance avoids the addition of a mineral acid, where, upon conversionof glycolonitrile to glycolic acid, the presence of a mineral acidand/or the production of the corresponding mineral acid salt may requirea purification step to remove the mineral acid and/or the correspondingsalt from the glycolic acid product. The pH of the acid-stabilizedglycolonitrile solution is typically adjusted with a base to a moreneutral pH range (i.e. pH of about 6 to about 8) prior to enzymaticconversion of glycolonitrile to glycolic acid (typically in the form ofthe ammonium salt of glycolic acid).

Acidovorax facilis 72W (ATCC 55746) Nitrilase

The A. facilis 72W nitrilase (EC 3.5.5.1) is a robust catalyst forproducing carboxylic acids from aliphatic or aromatic nitriles (WO01/75077; U.S. Pat. No. 6,870,038; and Chauhan et al., supra). It hasalso been shown to catalyze the conversion of α-hydroxynitriles (i.e.,glycolonitrile) to α-hydroxycarboxylic acids (i.e., glycolic acid) (seeU.S. Pat. No. 6,383,786 and U.S. Pat. No. 6,416,980). However, nitrilasecatalysts having improved nitrilase activity and/or stability (relativeto the A. facilis 72W nitrilase) when converting glycolonitrile toglycolic acid would reduce the cost of manufacturing glycolic acid. Assuch, a method of producing glycolic acid using an improved nitrilasecatalyst is needed to reduce the cost of manufacturing glycolic acid.

All known nitrilases, including the A. facilis 72W nitrilase, have anucleophilic cysteine in the enzyme active site (Cowan et al.,Extremophiles, 2:207-216 (1998); Pace, H. and Brenner, C., GenomeBiology, 2(1): reviews 1-9 (2001); and Chauhan et al., supra) and allare susceptible to inactivation by thiol reagents (1.0 mM concentrationsof copper chloride, silver nitrate, mercuric acetate, or ferric chlorideeach produced major decreases in A. facilis 72W nitrilase enzymeactivity). Cysteine residues are also capable of being irreversiblyoxidized to sulfinic acids, resulting in a loss of enzyme activity.Despite the sensitivity of nitrilase enzymes to various inactivatingmechanisms, immobilized A. facilis 72W cells are robust, capable ofretaining much of their nitrilase activity after numerous recyclereactions (U.S. Pat. No. 6,870,038).

Sequence comparisons of the A. facilis 72W nitrilase to other bacterialnitrilases have been reported (U.S. Pat. No. 6,870,038; Chauhan et al.,supra). The 72W nitrilase has several conserved signature domainsincluding a 16-amino acid region near the amino terminus (amino acidresidues 40-55 of SEQ ID NO:6) and the catalytic region (SEQ ID NO: 39;see amino acid residues 160-173 of SEQ ID NO:6) containing the essentialcysteine residue. This cysteine residue (Cys164 of SEQ ID NO:6), alongwith conserved glutamic acid (Glu48 of SEQ ID NO:6) and lysine residues(Lys130 of SEQ ID NO:6), form the catalytic triad motif found in allnitrilases (Pace, H., and Brenner, C., supra).

Nitrilase Enzymes Providing Improved Nitrilase Activity

Several mutant nitrilases (polypeptides having nitrilase activity)derived from the A. facilis 72W nitrilase have been previously reported(U.S. Ser. No. 10/919,182; herein incorporated by reference). In U.S.Ser. No. 10/919,182, various mutant nitrilases were selected andscreened for relative improvements (relative to the activity ofrecombinantly expressed, native 72W nitrilase) in nitrilase activity forconverting 3-hydroxynitriles to 3-hydroxyacids.

The expression system used with the nitrilase mutants described in U.S.Ser. No. 10/919,182 is based on the plasmid pTrcHis2-TOPO® and the E.coli host TOP10 (both from Invitrogen, La Jolla, Calif.). The activityof nitrilase mutants F168L (residue 168 changed from Phe to Leu in SEQID NO: 6), F168V (residue 168 changed from Phe to Val; SEQ ID NO: 32),F168K (residue 168 changed from Phe to Lys; SEQ ID NO: 26), T210A(residue 210 changed form Thr to Ala; SEQ ID NO: 34), and T210C (residue210 change from Thr to Cys; SEQ ID NO: 36) were compared to the nativeenzyme (“control”; SEQ ID NO: 6) in the same expression system using themethod described in Example 12. Mutants F168L, T210A, and T210C, whichwere initially identified as possibly having significantly improvednitrilase activity when converting GLN to GLA, were later found to havesimilar nitrilase activity to the 72W nitrilase control. Unexpectedly,two of the mutant nitrilases (F168K, Phe168→Lys; F168V, Phe168→Val)described in U.S. Ser. No. 10/919,182 (herein represented by SEQ ID NOs:26 and 32; respectively) also exhibited a significant improvement innitrilase activity when converting glycolonitrile (a 2-hydroxynitrile)to glycolic acid. However, the other mutant nitrilases described in U.S.Ser. No. 10/919,182 (e.g. T210A, SEQ ID NO: 34; T210C, SEQ ID NO: 36)did not show a significant improvement in nitrilase activity whenconverting glycolonitrile to glycolic acid.

As described in Examples 13-17 of the present application and incopending U.S. Provisional Application 60/638,176 (herein incorporatedby reference), error-prone PCR and targeted saturation mutagenesis wasused to randomly mutate the native 72W nitrilase coding sequence (SEQ IDNO: 5). Mutations that resulted in an amino acid substitution at aminoacid residue positions 168 (phenylalanine in the wild type sequence) and201 (leucine in the wild type sequence) appeared to increase nitrilaseactivity significantly (Examples 15-17). As used herein, the term “aminoacid residue position” refers to the amino acid found at a particularlocation relative to the reference sequence (SEQ ID NO: 6) as measuredfrom the N-terminal methionine residue. Targeted saturation mutagenesiswas conducted to evaluate all amino acid substitutions at both residuepositions (168 and 201). Several additional mutants were identifiedhaving a significant improvement nitrilase activity of convertingglycolonitrile to glycolic acid (e.g. in the form of the ammoniumglycolate salt under the present reaction conditions). In oneembodiment, suitable nitrilases useful in the present process comprise anucleotide sequence encoding a polypeptide having an amino acid of SEQID NO: 6 with at least one mutation that results in at least one aminoacid substitution selected from the group consisting of:

-   -   a) a substitution of lysine, methionine, threonine, or valine at        amino acid position 168; and    -   b) a substitution of glutamine, glycine, histidine, lysine,        asparagine, serine, alanine, cysteine, or threonine at amine        acid position 201; wherein said mutant has at least a 1.5-fold        improvement in nitrilase activity when converting glycolonitrile        to glycolic acid.

In another embodiment, suitable mutant nitrilase useful in the presentprocess have an amino acid sequence selected from the group consistingof SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32. Inyet another embodiment, suitable mutant nitrilase useful in the presentprocess have a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and 31.

Nitrilase activity was calculated by dividing the measured units ofactivity (U) by catalyst weight. The catalyst weight can be measured interms of purified protein weight, wet cell weight, dry cell weight orweight of the immobilized catalyst (i.e., using carrageenan and/orGA/PEI-crosslinked catalyst/alginate beads). In the present invention,the nitrilase activity was reported as units of activity per gram of drycell weight (U/g DCW) or units of activity per gram of catalyst bead(immobilized catalyst comparisons). With nitrilase activity comparisonsbased on dry cell weight as the unit catalyst weight, the level ofnitrilase protein production must be considered. The expression levelsof nitrilase enzyme between the various transformants and theirrespective controls were measured and observed to be essentiallyidentical (i.e., when compared in the same genetic background). Thus,improvements in the reported nitrilase activity for the various mutantswere attributed to structural modifications to the enzymes.

The coding sequences of the mutant nitrilases (and also of the A.facilis 72W (ATCC 55746) nitrilase control) were expressed in identicalvector backgrounds (pTrcHis2-TOPO® or pUC1 g) and hosts: E. coli TOP10(Invitrogen), E. coli FM5 (ATCC 53911), or E. coli MG1655 (ATCC 47076).Relative improvements were based on comparisons to the appropriatecontrol using the same vector and host background. SDS-PAGE analyses (asquantified using laser densitometry) demonstrated that nitrilase proteinexpression levels in each mutant (and control) were essentially equal(as expected due to the identical expression system and host used). Therelative enzyme activity was reported as the fold increase in nitrilaseactivity measured for the various mutant catalysts relative to thenitrilase activity measured in the respective E. coli controltransformant expressing the A. facilis 72W nitrilase (SEQ ID NO: 6).

For unimmobilized catalysts, nitrilase activity of the mutant nitrilases(U/g dry cell weight) was determined by measuring the rate of conversionof glycolonitrile to glycolic acid (μmol GLA/min) at 25° C. per gram ofdry cell weight. For immobilized catalyst comparisons, activity wasdetermined by measuring the rate of conversion of glycolonitrile toglycolic acid (μmol GLA/min) at 25° C. and reported as units ofnitrilase activity per gram of immobilized cell catalyst bead (U/gbead). One unit of nitrilase activity (U) is equivalent to production of1 micromole glycolic acid/min at 25° C. (μmol GLA/min).

For a particular mutant nitrilase, point substitution mutations withinthe DNA coding region and the resulting amino acid change are specifiedwith reference to the Acidovorax facilis 72W amino acid sequence (SEQ IDNO: 6), using one of the following formats:

-   -   1. Extended format: the wild-type amino acid is provided (using        the standard 3-letter abbreviation) along with the corresponding        amino acid residue position within SEQ ID NO:6 followed by the        new amino acid found within the mutant at the same residue        position. For example, “Phe168 to Lys” or “Phe168→Lys” describes        a mutation in the SEQ ID NO:6 at amino acid residue position 168        where phenylalanine was changed to lysine as a result of the        mutation.    -   2. Short-hand format: the wild-type amino acid (denoted by the        standard single letter abbreviation) is followed by the amino        acid residue position of SEQ ID NO:6 followed by the mutant        amino acid (also denoted by the standard single letter        abbreviation). For example, “F168K” describes a mutation in SEQ        ID NO: 6 at amino acid residue position 168 where phenylalanine        was changed to lysine as a result of the mutation.

Hydrolysis of Glycolonitrile to Glycolic Acid Using a Nitrilase Catalyst

The enzymatic conversion of glycolonitrile to glycolic acid (in the formof the acid and/or the corresponding ammonium salt) was performed bycontacting an enzyme catalyst (comprising a polypeptide having nitrilaseactivity) with a suitable aqueous reaction mixture comprisingglycolonitrile using a suitable set of enzymatic reaction conditions (pHrange, temperatures, concentrations, etc.) described below. In oneembodiment, whole recombinant microbial cells can be used as an enzymecatalyst without any pretreatment. In another embodiment, the microbialcell catalyst can be added directly to a reaction mixture, or maintainedseparately from the bulk reaction mixture using hollow-fiber membranecartridges or ultrafiltration membranes. In a further embodiment, themicrobial cells can be immobilized in a polymer matrix (e.g.,carrageenan or polyacrylamide gel (PAG) particles) or on an insolublesolid support (e.g., celite) to facilitate recovery and reuse of theenzyme catalyst (U.S. Pat. No. 6,870,038; herein incorporated byreference). In yet a further embodiment, purified or partially-purifiedenzyme(s) can also be isolated from the whole cells and used directly asa catalyst, or the enzyme(s) can be immobilized in a polymer matrix oron an insoluble support. Methods for the immobilization of cells or forthe isolated enzymes have been widely reported and are well known tothose skilled in the art (Methods in Biotechnology, Vol. 1:Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor;Humana Press, Totowa, N.J., USA; 1997). The immobilization of the A.facilis 72W nitrilase catalyst has previously been reported (U.S. Pat.No. 6,870,038).

The concentration of enzyme catalyst in the aqueous reaction mixturedepends on the specific activity of the enzyme catalyst and is chosen toobtain the desired rate of reaction. The wet cell weight of themicrobial cells used as catalyst in hydrolysis reactions typicallyranges from 0.001 grams to 0.250 grams of wet cells per mL of totalreaction volume, preferably from 0.002 grams to 0.050 grams of wet cellsper mL.

The temperature of the glycolonitrile hydrolysis reaction is chosen tocontrol both the reaction rate and the stability of the enzyme catalystactivity. The temperature of the reaction may range from just above thefreezing point of the reaction mixture (approximately 0° C.) to about65° C., with a preferred range of reaction temperature of from about 5°C. to about 35° C. The microbial cell catalyst suspension may beprepared by suspending the cells in distilled water, or in a aqueoussolution of a buffer which will maintain the initial pH of the reactionbetween about 5.0 and about 10.0, preferably between about 5.5 and about8.0, more preferably between about 5.5 and about 7.7, and mostpreferably about 6.0 to about 7.7. As the reaction proceeds, the pH ofthe reaction mixture may change due to the formation of an ammonium saltof the carboxylic acid from the corresponding nitrile functionality. Thereaction can be run to complete conversion of glycolonitrile with no pHcontrol, or a suitable acid or base can be added over the course of thereaction to maintain the desired pH.

Glycolonitrile was found to be completely miscible with water in allproportions at 25° C. In cases where reaction conditions are chosen suchthat the solubility of the substrate (i.e., an α-hydroxynitrile) is alsodependent on the temperature of the solution and/or the saltconcentration (buffer or product glycolic acid ammonium salt, also knownas ammonium glycolate) in the aqueous phase, the reaction mixture mayinitially be composed of two phases: an aqueous phase containing theenzyme catalyst and dissolved α-hydroxynitrile, and an organic phase(the undissolved α-hydroxynitrile). As the reaction progresses, theα-hydroxynitrile dissolves into the aqueous phase, and eventually asingle phase product mixture is obtained. The reaction may also be runby adding the α-hydroxynitrile to the reaction mixture at a rateapproximately equal to the enzymatic hydrolysis reaction rate, therebymaintaining a single-phase aqueous reaction mixture, and avoiding thepotential problem of substrate inhibition of the enzyme at high startingmaterial concentrations.

Glycolic acid may exist in the product mixture as a mixture of theprotonated carboxylic acid and/or its corresponding ammonium salt(dependent on the pH of the product mixture; pKa of glycolic acid isabout 3.83), and may additionally be present as a salt of the carboxylicacid with any buffer that may additionally be present in the productmixture. Typically, the glycolic acid produced is primarily in the formof the ammonium salt (pH of the glycolonitrile hydrolysis reaction istypically between about 5.5 and about 7.7). The glycolic acid productmay be isolated from the reaction mixture as the protonated carboxylicacid, or as a salt of the carboxylic acid, as desired.

The final concentration of glycolic acid in the product mixture atcomplete conversion of glycolonitrile may range from 0.001 M to thesolubility limit of the glycolic acid product. In one embodiment, theconcentration of glycolic acid will range from about 0.10 M to about 5.0M. In another embodiment, the concentration of glycolic acid will rangefrom about 0.2 M to about 3.0 M.

Glycolic acid may be recovered in the form of the acid or correspondingsalt using a variety of techniques including, but not limited to ionexchange (Example 38), electrodialysis, reactive solvent extraction(Examples 39-61), polymerization, thermal decomposition (Examples62-66), alcoholysis (Examples 67-74), and combinations thereof.

Microbial Expression

The present nitrilase mutants may be produced in heterologous hostcells, preferably in microbial hosts. Particularly useful in the presentinvention will be cells that are readily adaptable to large-scalefermentation methods. Such organisms are well known in the art ofindustrial bioprocessing, examples of which may be found in RecombinantMicrobes for Industrial and Agricultural Applications, Murooka et al.,eds., Marcel Dekker, Inc., New York, N.Y. (1994), and includefermentative bacteria as well as yeast and filamentous fungi. Host cellsmay include, but are not limited to Comamonas sp., Corynebacterium sp.,Brevibacterium sp., Rhodococcus sp., Azotobacter sp., Citrobacter sp.,Enterobacter sp., Clostridium sp., Klebsiella sp., Salmonella sp.,Lactobacillus sp., Aspergillus sp., Saccharomyces sp., Zygosaccharomycessp., Pichia sp., Kluyveromyces sp., Candida sp., Hansenula sp.,Dunaliella sp., Debaryomyces sp., Mucor sp., Torulopsis sp.,Methylobacteria sp., Bacillus sp., Escherichia sp., Pseudomonas sp.,Rhizobium sp., and Streptomyces sp. Particularly preferred is E. coli.Examples of suitable E. coli host cells in which a mutant nitrilase genecan be expressed include, but are not limited to, host cells specifiedherein and MG1655 (ATCC 47076), FM5 (ATCC 53911), W3110 (ATCC 27325),MC4100 (ATCC 35695), W1485 (ATCC 12435), and their derivatives. Inanother aspect, the preferred E. coli host strains are MG1655 (ATCC47076) or FM5 (ATCC 53911),

Heterologous expression of the A. facilis 72W nitrilase has previouslybeen reported (Chauhan et al., supra and U.S. Pat. No. 6,870,038).Chauhan et al. report an E. coli strain (E. coli SS1001 (ATCC PTA-1177))that expressed active A. facilis 72W nitrilase (SEQ ID NO: 38). Thecoding sequence of the recombinantly expressed (E. coli SS1001)nitrilase contained two minor sequence changes in comparison to thewild-type 72W nitrilase sequence (SEQ ID NOs: 5 and 6). The start codonwas changed from GTG to ATG to facilitate recombinant expression and anartifact was introduced during cloning that resulted in a single aminoacid change near the C-terminal (Pro367 [CCA]→Ser [TCA]).

Recombinant expression in an industrially-suitable host has severaladvantages. First, the genetic toolbox for many of the commonly usedproduction hosts is usually well developed in comparison to the genetictools available for many of the microorganisms from which the gene ofinterest was obtained. Recombinant expression in these hosts is normallymore cost effective than expression in the native host. For example, ithas been shown that A. facilis 72W cells grow on glycerol, a relativelyexpensive carbon substrate, when grown by fermentation, and have notbeen successfully grown using inexpensive glucose. In contrast, E. colitransformants can be grown on glucose to the same cell density as A.facilis 72W cells in about half the time, significantly reducingbiocatalyst production costs (U.S. Pat. No. 6,870,038).

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well-known to those skilled in the art. These could be usedto construct chimeric genes for production of the gene products of thepresent mutant nitrilases. These chimeric genes could then be introducedinto appropriate microorganisms via transformation to provide high levelexpression of the mutant nitrilase enzymes. The nucleic acid moleculesof the present invention are used to produce gene products havingenhanced or altered nitrilase activity levels relative to that of thenative A. facilis 72W nitrilase. In one aspect, the polypeptides encodedby the present mutant genes provide at least a 1.5 fold improvement innitrilase activity (as compared to the activity of the A. facilis 72Wnitrilase “control” represented by SEQ ID NO: 6) for convertingglycolonitrile to glycolic acid.

Chimeric genes will be effective in altering the properties of a hostcell. For example, introducing at least one copy of chimeric genesencoding the present nitrilases under the control of the appropriatepromoters into a host cell gives the host cell an improved ability toconvert glycolonitrile to glycolic acid. The chimeric genes willcomprise suitable regulatory sequences useful for driving geneexpression of the present mutant nitrilase sequences. Suitableregulatory sequences may include, but are not limited to promoters,translation leader sequences, and ribosomal binding sites. It ispreferred if these sequences are derived from the host organism;however, the skilled person will recognize that heterologous regulatorysequences may also be used.

Chimeric genes can be introduced into an appropriate host by cloning itinto a suitable expression vector. Vectors or cassettes useful for thetransformation of suitable host cells are well known in the art.Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the codingsequence that harbors transcriptional initiation controls and a region3′ of the DNA fragment which controls transcriptional termination. It ismost preferred when both control regions are derived from geneshomologous to the host cell, although such control regions need not bederived from the genes native to the specific species chosen as aproduction host.

In one embodiment, the regulatory sequences will include a promoter.Promoters may be constitutive or inducible. Inducible promoters aregenerally responsive to a specific stimulus (e.g., IPTG or lactoseinducing the lac promoter). Inducible promoters may be responsive to avariety of stimuli, including, chemicals, growth cycle, changes intemperature, changes in pH and changes in osmolarity, to name only afew.

Initiation control regions or promoters that are useful to driveexpression of the present mutant nitrilases in the desired host cell arenumerous and familiar to those skilled in the art, including but notlimited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1,URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1(useful for expression in Pichia); and lac, trp, IP_(L), IP_(R), T7,tac, P_(BAD), npr, and trc (particularly useful for expression inEscherichia coli). Additional examples of promoters particularlysuitable for driving expression in E. coli include, but are not limitedto the tryptophan operon promoter Ptrp of E. coli, a lactose operonpromoter Plac of E. coli, a Ptac promoter of E. coli, a phage lambdaright promoter P_(R), a phage lambda left promoter P_(L), a T7 promoter,and a promoter of the GAP gene from Pichia pastoris, or is at least onepromoter isolated from the group of microorganisms selected from thegroup consisting of Comamonas, Corynebacterium, Brevibacterium,Rhodococcus, Azotobacter, Citrobacter, Enterobacter, Clostridium,Klebsiella, Salmonella, Lactobacillus, Aspergillus, Saccharomyces,Pichia, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula,Dunaliella, Debaryomyces, Mucor, Torulopsis, Methylobacteria, Bacillus,Escherichia, Pseudomonas, Rhizobium, and Streptomyces.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary; however, it is most preferred if included.

Additionally, the inserted genetic material may include a ribosomebinding site (RBS). The ribosome binding site may be from a phage lambdaCII gene or is selected from the group consisting of ribosome bindingsites from a gene of Comamonas, Corynebacterium, Brevibacterium,Rhodococcus, Azotobacter, Citrobacter, Enterobacter, Clostridium,Klebsiella, Salmonella, Lactobacillus, Aspergillus, Saccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Dunaliella, Debaryomyces, Mucor, Torulopsis, Methylobacteria, Bacillus,Escherichia, Pseudomonas, Rhizobium, and Streptomyces.

Optionally, the gene products may preferably be a secreted product ofthe transformed host. Secretion of desired proteins into the growthmedia simplifies purification procedures and reduces costs. Secretionsignal sequences are often useful in facilitating the active transportof expressible proteins across cell membranes. A transformed hostcapable of secretion may be created by incorporating in the host a DNAsequence that codes for a secretion signal. Methods for choosingappropriate signal sequences are well known in the art (see for exampleEP 546049; WO 93/24631). The secretion signal DNA may be located betweenthe expression-controlling DNA and the instant coding sequence or codingsequence fragment, and in reading frame with the latter.

Method to Improve Nitrilase Stability and Productivity when ConvertingGlycolonitrile to Glycolic Acid

Stabilizing Agents

Glycolonitrile can be synthesized by reacting formaldehyde with hydrogencyanide (U.S. Pat. No. 2,175,805, U.S. Pat. No. 2,890,238, U.S. Pat. No.5,187,301, and copending U.S. Provisional Patent Application60/638,127). Depending upon the purity of the reactants and the reactionconditions used to make glycolonitrile, a variety of impurities mayexist in the final product. These impurities can interfere with theefficiency of converting glycolonitrile to glycolic acid. In oneembodiment, the aqueous glycolonitrile solution may be optionallytreated to remove undesirable impurities prior to being enzymaticallyconverted to glycolic acid. In a preferred embodiment, theglycolonitrile produced in the present process does not require anyadditional purification steps prior to contact with the present enzymecatalysts.

Another method to increase the stability/productivity of an enzymecatalyst is the addition of one or more compounds that will react withundesirable compounds in the glycolonitrile solution that may interferewith the catalyst stability and/or productivity (See copending U.S.Patent Application 60/638,176; herein incorporated by reference). Theundesirable compounds include, but are not limited to formaldehyde,formaldehyde-derived impurities, formaldehyde-derived oligomers andpolymers, glycolamide, glycolamide-derived impurities, hydrogencyanide-derived impurities, hydrogen cyanide-derived oligomers andpolymers, glycolonitrile-derived impurities, glycolonitrile-derivedoligomers and polymers, glycolide, linear oligomers of glycolic acid,and possibly oxygen (reactions conducted under substantially oxygen freeconditions improved catalyst stability). The undesirable compounds mayalso include those that 1) react with and inactivate the nitrilasecatalyst, 2) compete with glycolonitrile in the reaction, 3) react withglycolonitrile or glycolic acid to form unwanted byproducts, or 4)adversely affect the recombinant host cell (i.e. promote cell lysis).Examples of suitable compounds that can be added to the glycolonitrilereaction mixture may include, but are not limited to thiosulfates (e.g.potassium thiosulfate, K₂S₂O₃), dithionites (e.g., sodium dithionite,Na₂S₂O₄), and cyanide compounds (e.g. HCN, NaCN, KCN, etc.). In oneembodiment, the compound is added to the glycolonitrile reaction mixturebefore, during, or after the addition of the enzyme catalyst. In anotherembodiment, the compound is added to the reaction mixture so that thefinal concentration in the reaction mixture is at least about 0.001 M toless than about 5 wt % of the reaction mixture. In a further embodiment,the compound is added to the reaction mixture so that the finalconcentration is at least about 0.01 M. In yet a further embodiment, thecompound is added to the reaction mixture so that the finalconcentration of compound is about 0.01 M to about 1 M.

In a further aspect of the invention, the present process is conductedunder substantially oxygen free conditions. As used herein, the terms“oxygen free conditions”, “oxygen free atmosphere” and “substantiallyoxygen free conditions” refers to a reaction condition where anon-reactive gas, such as nitrogen, is used to purge and/or blanket thereaction vessel so that molecular oxygen (O₂) is not present duringpresent process. One of skill in the art recognizes that trace amountsof molecular oxygen may exist under substantially oxygen freeconditions. In one aspect, the term “substantially oxygen free” means areaction condition where the molecular oxygen concentration is less thanabout 5%, preferably less than 2%, more preferably less than 1%, andmost preferably less than 0.1% of the gas in the reaction vessel. Inanother aspect, the present process is conducted under substantiallyoxygen free conditions where nitrogen (N₂) is used to blanket theaqueous reaction mixture in the reaction vessel.

Controlling the Concentration of Glycolonitrile

Another method to increase the nitrilase stability is controlling themaximum concentration of glycolonitrile in the aqueous reaction mixture(U.S. 60/638,176). As described previously, glycolonitrile dissociatesin polar solvents to release formaldehyde and hydrogen cyanide.Formaldehyde in the reaction mixture may react with the enzyme catalystleading to premature inactivation and a decrease in catalystproductivity. Controlling the concentration of the glycolonitrile insolution can increase both the catalyst stability and productivity ofthe catalyst (grams of glycolic acid produced per gram of catalyst). Asshown in Examples 22-25 (Table 10), a nitrilase catalyst derived fromthe Acidovorax facilis 72W rapidly loses its activity in reactions thatcontain 3 M glycolonitrile after only 3 recycle reactions. Decreasingthe concentration to 1 M and/or the stepwise addition of 3 Mglycolonitrile in 1 M increments (added after the previous additions ofglycolonitrile have been converted to ammonium glycolate) increases thecatalyst productivity significantly (Table 10). In one embodiment, astepwise addition (aliquots) of glycolonitrile to the aqueous reactionmixture increases the productivity of the catalyst. In anotherembodiment, the glycolonitrile is added to the aqueous reaction mixturein a stepwise fashion so that the total concentration of glycolonitrileremains about 1 M or less during the reaction.

As shown in Example 25, a continuous addition of glycolonitrile alsoincreases the catalyst productivity over several recycle reactions. Inone embodiment, the method to produce ammonium glycolate fromglycolonitrile uses a continuous addition of glycolonitrile. In anotherembodiment, the rate of glycolonitrile addition is at least 5-times theKm of the catalyst. The present catalysts typically have a Km forglycolonitrile of approximately 1 mM (wild type A. facilis 72W; SEQ IDNO: 6) to about 2.7 mM. As known in the art, a substrate concentrationof approximately 5-times the Km value (i.e., 5×2.7 mM=13.5 mM) resultsin a reaction rate that is approximately 97% of the maximum reactionrate (Vmax). In yet another embodiment, the glycolonitrile feed rate iscontrolled to maintain a glycolonitrile concentration in the reactionmixture of about 5 mM to about 1 M, preferably about 100 mM to about 1M, more preferably about 100 mM to about 500 mM.

Control of pH

Reactions using the nitrilase catalysts of the present process aretypically run at a pH ranging from about 5 to about 10, preferablybetween 5.5 and about 8, more preferably about 5.5 to about 7.7, andmost preferably about 6 to about 7.7.

Industrial Production of the Microbial Catalyst

Where commercial production of the present nitrilases using the presentmutated nitrilase genes is desired, a variety of culture methodologiesmay be used. Fermentation runs may be conducted in batch, fed-batch, orcontinuous mode, methods well-known in the art (Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., (1989); Deshpande,Mukund V., Appl. Biochem. Biotechnol. 36(3): 227-234 (1992)).

A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media. Measurement of the actual substrateconcentration in Fed-Batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen, and the partial pressure of waste gases such as CO₂.Batch and Fed-Batch culturing methods are common and well known in theart and examples may be found in Brock (supra) and Deshpande (supra).

Commercial production of the present nitrilase catalysts may also beaccomplished with a continuous culture. Continuous cultures are an opensystem where a defined culture media is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high-liquid-phase density where cells areprimarily in log phase growth. Alternatively, continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productsare continuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

Continuous or semi-continuous culture allows for the modulation of onefactor or any number of factors that affect cell growth or end cellconcentration. For example, one method will maintain a limiting nutrientsuch as the carbon source or nitrogen level at a fixed rate and allowall other parameters to moderate. In other systems a number of factorsaffecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady-state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of cell formation, are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock(supra).

Fermentation media in the present invention must contain suitable carbonsubstrates. Suitable substrates may include, but are not limited tomonosaccharides such as glucose and fructose, disaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof, and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Hence, it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by the choice oforganism.

Analysis of Glycolic Acid and Glycolonitrile

Analytical methods suitable for analyzing the production of glycolicacid are well-known in the art including, but not limited to ¹H NMR, ¹³CNMR, HPLC, CE, GC, and MS. For example, HPLC analysis was used todetermine the amount of glycolic acid production using a refractiveindex detector and a Bio-Rad HPX-87H column (30 cm×7.8 mm dia.) and 0.01N sulfuric acid at 1.0 mL/min (isocratic) as a mobile phase at 50° C.The HPLC method was suitable for quantification of both the substrate(glycolonitrile) and product (glycolic acid).

Methods to Recover Glycolic Acid from Ammonium Glycolate

There are many methods that can be used to recover/isolate anα-hydroxyacid (i.e., glycolic acid) from an aqueous solution comprisingammonium glycolate including, but not limited to ion exchange (anionicand/or cationic), electrodialysis, reactive solvent extraction,alcoholysis (esterification followed by hydrolysis of the glycolic acidester into glycolic acid), thermal salt cracking, and combinationsthereof.

Ion Exchange (Cationic)

Cationic ion exchange is a reversible process is which a dissolved ionicspecies is taken up by a solid in a stoichiometric manner. Cationic ionexchange is well known in the art. In the present process, ammoniumglycolate is fed to the cationic ion exchange resin where the ammoniumion is exchanged with a proton, forming glycolic acid (see Example 38).The glycolic acid passes through the column and is collected.

Once the resin is saturated with ammonium ion, regeneration with anacid, for example sulfuric acid, will produce the byproduct, ammoniumsulfate salt. The cationic exchange can be performed in batches, using asimulated moving bed or carrousel (see Perry's Chemical Engineers'Handbook, 7^(th) ed., Perry, Robert H., Green, Dow W., and Maloney,James O., editors; McGraw Hill Companies, Inc., New York, N.Y., 1997;hereinafter “Perry's”). Selection of the resin may impact feedconcentration, which may range from about 0.02 wt % salt to about 50 wt% ammonium glycolate, preferably about 0.02 wt % to about 40 wt %. Theregeneration acid used typically ranges from about 0.5 wt % to about 20wt %.

Ion Exchange (Anionic)

Anionic ion exchange is also well known in the art. Anionic exchange issimilar to cationic exchange except that a weak anionic resin is used(see Perry's, supra). Once again, selection of the resin may impact feedconcentration, which may range from about 0.02 wt % to about 90 wt %ammonium glycolate, preferably about 0.02 wt % to about 40 wt %.Regeneration of the resin would typically use a weak acid.

Solvent Extraction (Reactive)

One method that has been used to isolate carboxylic acids is reactiveextraction. This method has been reported to be useful for extractinglactic acid from ammonium lactate (Wasewar et al., J. Biotechnol.,97:59-68 (2002)). Reactive extraction involves the use of a reactiveorganic solvent (i.e., an amine) to complex with the acid in the aqueousphase. The first step in the process typically involves acidification ofthe aqueous solution containing the salt of the desired acid. Theacidified aqueous solution is then contacted with an organic solventtypically comprised of a reactive tertiary amine and one or morediluents. The reactive amine (typically a tertiary C8-C10 trialkylaminesuch as Alamine® 336, Cognis Corp, Cincinnati, Ohio) reacts with thecarboxylic acid forming an acid/amine complex that is preferentiallysoluble in the organic phase (Tamada et al., Ind. Eng. Chem. Res.29:1319-1326 (1990); Tamada et al., Ind. Eng. Chem. Res. 29:1327-1333(1990)). The use of a tertiary alkylamine typically provides much higherdistribution coefficients than would be obtainable with normal solventextraction. Back extraction is then used to recover the acid from theorganic phase.

Inci, I. (Chem. Biochem. Eng. Q., 16(2):81-85 (2002); Inci, I. and Uslu,H., J. Chem. Eng. Data, 50:536-540 (2005)) report the use of reactiveamine solvents for the extraction of glycolic acid. However, theseexperiments reported the extraction coefficients of pure glycolic aciddissolved in pure water. Inci does not illustrate or teach a process toobtain glycolic acid from a complex aqueous matrix (e.g., aqueoussolutions of glycolic acid comprising significant amounts of mineralsalts and other impurities), such as concentrated aqueous solutions ofammonium glycolate.

Reactive solvent extraction may also be used to obtain glycolic acidfrom an aqueous solution of ammonium glycolate (see present Examples39-61 and Co-pending U.S. provisional patent application 60/638,128;herein incorporated by reference). More specifically, a method toisolate glycolic acid from an aqueous solution comprising ammoniumglycolate is provided comprising:

-   -   a) providing a first phase, wherein said first phase is a        water-immiscible organic solvent mixture comprising:        -   i) about 30 volume percent to about 99 volume percent of            said first phase is at least one tertiary alkyl amine having            the formula

-   -   -   -   wherein R₁, R₂, and R₃ are independently a C8 to C12                alkyl group; and

        -   ii) about 1 volume percent to about 70 volume percent of            said first phase is at least one diluent selected from the            group consisting of methyl isobutyl ketone, 1-octanol,            1-decanol, methylene chloride, 1-chlorobutane,            chlorobenzene, chloroform, kerosene, toluene, mixed xylenes,            tributyl phosphate, and mixtures thereof;

    -   b) providing a second phase, wherein said second phase is an        aqueous solution comprising glycolic acid having a pH of about 3        or less; said second phase formed by the process of:        -   i) providing an aqueous solution of ammonium glycolate; said            aqueous solution of ammonium glycolate having a            concentration about 5 weight % to about 40 weight % ammonium            glycolate; and        -   ii) adding an amount of mineral acid sufficient to lower the            pH of the aqueous ammonium glycolate solution of (b)(i) to            about 3 or less; whereby an aqueous solution comprising            glycolic acid is formed;

    -   c) contacting said first phase with said second phase in a        reactive extraction process; thereby forming a glycolic        acid-loaded first phase;

    -   d) isolating said glycolic acid-loaded first phase;

    -   e) contacting said glycolic acid-loaded first phase with a third        phase in a back extraction process; whereby glycolic acid in the        glycolic acid-loaded first phase is extracted into said third        phase; wherein said third phase is an aqueous solution that is        immiscible in said glycolic acid-loaded first phase; and

    -   f) recovering the glycolic acid from said third phase.

In one embodiment, the tertiary trialkylamine is selected from the groupconsisting of tri-n-octylamine, tri-isooctylamine, tri-n-nonylamine,tri-n-decylamine, and tri-n-dodecylamine. In another embodiment, thetertiary trialkylamine is selected from the group consisting of Alamine®308 (CAS# 2757-28-0), Alamine® 300 (CAS# 1116-76-3), Alamine® 304-1(CAS# 102-87-4), and Alamine® 336 (CAS# 68814-95-9) (Cognis Corp.,Cincinnati, Ohio). In a further embodiment, the diluent is selected fromthe group consisting of methyl isobutyl ketone (MIBK), kerosene,toluene, mixed xylenes, 1-octanol, and mixtures thereof. In yet anotherembodiment, the water-immiscible organic solvent is selected from thegroup consisting of 90% (vol/vol) Alamine® 336:10% (vol/vol) MIBK; 90%Alamine® 336:10% 1-octanol; 90% Alamine® 336:10% toluene; and 90%Alamine® 336:10% mixed xylenes.

The concentration of tertiary trialkyl amine in the first phase mayrange from about 30 percent (vol/vol) to about 99 percent (vol/vol),preferably about 50 percent (vol/vol) to about 90 percent (vol/vol), andmost preferably about 70 percent (vol/vol) to about 90 percent(vol/vol). The amount of diluent in the first phase may range from about1 percent (vol/vol) to about 70 percent (vol/vol), preferably about 10percent to about 50 percent, and most preferably about 10 to about 30percent

A suitable organic extraction mixture for extracting glycolic acid iscomprised of a mixture of Alamine® 336 with one or more diluentsselected from the group consisting of methyl isobutyl ketone (MIBK),1-octanol, 1-decanol, methylene chloride, 1-chlorobutane, chlorobenzene,chloroform, kerosene, toluene, mixed xylenes, and tributyl phosphate. Inone embodiment, the organic phase extractant is comprised of Alamine 336in combination with one or more diluents selected from the groupconsisting of MIBK, 1-octanol, toluene, xylene, and kerosene. In anotherembodiment, the reactive organic solvent is comprised of about 50% toabout 95% Alamine® 336, preferably about 65% to about 95% of the organicsolvent mixture. The organic solvent is comprised of one or morediluents in a range of about 50% to about 5% diluent, preferably 35% toabout 5% of the organic solvent mixture. In one embodiment, the mixedorganic solvent is comprised of about 70% Alamine® 336, about 10% MIBK,and about 20% kerosene. In another embodiment, the mixed organic solventis comprised of about 90% Alamine® 336 and about 10% diluent selectedfrom the group consisting of MIBK, 1-octanol, toluene, and xylene.

One of skill in the art can determine the preferred temperature of theorganic phase extraction. In one embodiment, the extraction reaction isconducted at a temperature from about 10° C. to about 90° C., morepreferably about 20° C. to about 75° C., and most preferably about 25°C. to about 75° C.

The amount of mixed organic solvent required to extract the glycolicacid from the acidified aqueous phase is dependent upon the degree ofsolvent loading. One of skill in the art can adjust the volume of themixed organic solvent used to extract the glycolic acid depending uponthe amount of glycolic acid present in the aqueous phase. The glycolicacid can be recovered from the organic phase by back extraction.

Another method to obtain glycolic acid from ammonium glycolate isthermal decomposition in the presence of an esterifying agent. Thesolvent may act by protecting the glycolic acid from reactive ammonia(thereby preventing amide formation) or may act as an organic reactiveextraction solvent, thereby aiding in the separation of the acid (Menget al., US 2004/0210087; hereby incorporated by reference in itsentirety). Optionally, this method can also include an alcohol, therebycreating the ester (which may be more soluble in the organic solvent).The organic solvent may be selected from the group consisting oftertiary alkylamines, alcohols, amides, ethers, ketones, phosphorusesters, phosphine oxides, phosphine sulfides, alkyl sulfides, andcombinations thereof. Glycolic acid (or the corresponding ester) is thenrecovered from the organic solvent (liquid phase) during a backextraction step. The recovered solvent can be recycled to the saltsplitting reaction step. Unfortunately, solvent extraction/backextraction may be problematic as various immiscible fluids form complexphysical mixtures that are difficult to separate.

Alcoholysis (Esterification)

Cockrem (U.S. Pat. No. 6,291,708 B1) teaches rapid heating of a mixtureof ammonium salt of an organic acid with alcohol to produce a liquidstream containing acid, ester, and unreacted ammonium salt. Cockremfails to address the separation of unreacted salts from the acid andester. However, a process of using alcoholysis (heated alcohol vaporthat acts as both an esterification agent and stripping gas) to separatethe glycolic acid ester (as a vapor) from the aqueous solutioncomprising ammonium glycolate has been described that addresses thisproblem (See present Examples 67-74 and copending U.S. Provisionalapplication 60/638,126; herein incorporated by reference).

In order to overcome the problems associated with separating an esterfrom unreacted ammonium glycolate in a liquid matrix, alcoholysis may beused (U.S. 60/638,126). The ammonium salt of a carboxylic acid (i.e.,ammonium glycolate) will react with alcohols to form an ester of thealcohol and acid while liberating ammonia with water as shown inEquation 4.

HOCH₂CO₂ ⁻NH₄ ⁺+R₂OH→HOCH₂CO₂R₂+NH₃+H₂0  (4)

U.S. 60/638,126 provides a process to obtain glycolic acid from anaqueous solution comprising ammonium glycolate comprising:

-   -   (a) providing        -   (i) an aqueous solution comprising ammonium glycolate; and        -   (ii) a heated alcohol vapor feed stream comprising an            alcohol having the formula:

R₂—OH

-   -   -   wherein R₂ is a C1 to C4 straight chain or branched alkyl            group; and        -   (iii) a reaction vessel;

    -   (b) contacting said aqueous solution comprising ammonium        glycolate with said heated alcohol vapor feed stream in said        reaction vessel whereby a first vapor product stream is produced        comprising a glycolic acid ester;

    -   (c) recovering the glycolic acid ester from said first vapor        product stream;

    -   (d) hydrolyzing the glycolic acid ester of (c) into glycolic        acid; and

    -   (e) recovering the glycolic acid produced in step (d).

In one embodiment, the alcohol is selected from the group consisting ofmethanol, ethanol, n-propanol, isopropanol, n-butanol, isobutyl alcohol,and t-butanol. In a preferred embodiment, the alcohol is methanol (i.e.,forming methyl glycolate in the vapor product stream).

The amount of heated alcohol vapor contacted with the carboxylic acidammonium salt is typically in a molar excess relative to the amount ofcarboxylic acid ammonium salt in the aqueous feed stream. The molarratio of the heated alcohol vapor to the carboxylic acid ammonium saltmay vary, but it typically from about 5 to about 300 moles per mole ofcarboxylic acid ammonium salt (molar ration at least about 5:1 to about300:1), preferably about 5 to about 200 moles per mole of the carboxylicacid ammonium salt, most preferably about 20 to about 100 moles per moleof carboxylic acid ammonium salt. A molar excess of the alcohol vaportends to inhibit amide formation.

The alcohol vapor feed stream (e.g., methanol) temperature is typicallychosen to ensure that the alcohol generally remains in its vapor phaseso that it acts as both an esterifying agent and a stripping/carryinggas. The temperature of the heated alcohol vapor feed stream enteringthe reaction chamber may vary according to the selected alcohol as wellas the specific equipment geometry. The heated alcohol vapor fed acts asa source of heat for the reaction, an esterifying agent, and as astripping/carrying gas for the carboxylic acid ester formed by thepresent process.

The present examples illustrate the use a heated methanol vapor to formmethyl glycolate (which is subsequently hydrolyzed to glycolic acid).Typically, the temperature of the heated methanol vapor is about 140° toabout 350° C. In one embodiment, the temperature of the methanol vaporfeed stream is about 170° C. to about 300° C. In another embodiment, thetemperature of the methanol vapor feed stream is about 230° C. to about250° C.

The reactor pressure and temperature can be adjusted to optimizeproduction of the desired product. Selecting the appropriate operatingtemperature and pressure for the reaction must consider the vaporpressure of both the alcohol and the corresponding carboxylic acidester. At the selected operating pressure, the reaction temperature isselected so that the vapor pressure of the carboxylic acid ester istypically at least about one quarter (¼) of the operating pressure ofthe system. At this temperature the vapor pressure of the alcohol shouldexert at least about 4 times (4×) the operating pressure. A typicaloperating pressure is from about 0 psig (˜0 kilopascals (kPa)) to about80 psig (˜550 kPa), preferably about 0 psig (0 kPa) to about 50 psig(345 kPa), and most preferably about 10 psig (69 kPa) to about 50 psig(345 kPa).

A typical operating temperature for the alcoholysis reactor is about140° C. to about 300° C., preferably about 170° C. to about 200° C. Inone aspect, the carboxylic acid ammonium salt is ammonium glycolate andthe alcohol is methanol. The reactor temperature used this particularcombination is typically about 100° C. to about 300° C., preferablyabout 150° C. to about 250° C., more preferably about 170° C. to about225° C., and most preferably about 170° C. to about 200° C.

The reactor may optionally include a packing material or a high boilingpoint fluid/liquid to improve the yield of the desired carboxylic acidester. The benefit of the packing or high boiling point fluid is toimprove the contacting between the aqueous salt solution and the alcoholvapor. The packing may be random packing, engineered packing, or variousdistillation plate designs. See Perry's 7^(th) edition Chapter 14.23through 14.61 (Perry's Chemical Engineers' Handbook, 7^(th) ed., Perry,Robert H., Green, Dow W., and Maloney, James O., editors; McGraw HillCompanies, Inc., New York, N.Y., 1997). Commercial designs for gasliquid reaction systems are illustrated in Perry's FIGS. 23-25, 23-26,and 13-79. The high boiling point fluid should be selected to have a lowvapor pressure at the chosen operating conditions or be easily separatedfrom the recovered ester. The fluid may be inert to the esterificationchemistry (such as mineral oil) or potentially participate in theesterification chemistry such as a polyol. The polyol is a material witha molecular weight greater than 150 and at least one hydroxyl group,including alcohols such as decanol and dodecanol. Typical polyolsinclude polyethylene ether glycol (PEG), polypropylene ether glycol(PPG) and polytetramethylene ether glycol (PTMEG), as well as copolymersof these polyalkylene ether glycols.

Recovering the ester as a liquid from the first vapor product may beaccomplished by reducing the temperature of the vapor to form acondensate. The cooling may be accomplished in a direct or indirectcontact condenser (see Perry's Chapter 11; supra). The condenser (“hotcondenser”) temperature is typically maintained at or below the boilingpoint of the respective carboxylic acid ester but above the normalboiling point of the heated alcohol vapor. Typically, the partialcondenser temperature is maintained at least about 10° C. to about 100°C. below the normal boiling point of the ester. Control of the alcoholvapor temperature, the reactor pressure, and the partial condensertemperatures should be used to selectively condense the desiredcarboxylic acid ester from the corresponding esterifying agent (i.e. thealcohol), water, and ammonia vapors.

Distillation may also be used to obtain the carboxylic acid ester fromthe vapor product stream. Distillation designs (e.g., generallycomprised of a reflux column, an overhead condenser, and reflux control)are well know. Commercial designs for distillation systems may be foundin Perry's Chapter 13. Designs with multiple product removal may beparticularly well suited for recovering the ester (See Perry's FIGS.13-6).

In one embodiment, the gas liquid contacting operation and the esterrecovery from the first vapor product operation may be accomplished in asingle device.

The corresponding carboxylic acid can be subsequently obtained byhydrolyzing the carboxylic acid ester collected in the first liquidproduct stream (i.e., from the partial condenser). Techniques tohydrolyze esters to acids are known to those skilled in the art. Therecovered ester can be combined with water and placed into a short pathbatch distillation apparatus containing a short fractionating column anda total condenser. Heating the mixture will drive methanol overhead aswell as some of the water, leaving the carboxylic acid behind in theheated mixture.

Electrodialysis

Electrodialysis with bipolar membrane (EDBM) has been proposed torecovering organic acids from their corresponding ammonium salt. ForEDBM to work, the solutions must be conductive. For the ammonium salt ofweak acids, the products of EDBM (organic acid and ammonium hydroxide)are very weak conductors resulting in high resistance of the solutionsand low production rates. To offset this, a conductive salt (i.e.,ammonium chloride) is added to the base loop (ammonium hydroxidestream). As the base concentration increases, ammonia can be strippedfrom the solution and the ammonium salt recycled to maintainconductivity.

The composition of the ammonium salt of the organic acid must becarefully monitored for multivalent cations such as calcium. Thesecations may precipitate by associating with hydroxyl groups and destroythe membranes. Concentrations of the multivalent cations are preferablybelow about 5 ppm, most preferably below about 1 ppm.

For example, a typical lab scale EDBM system can be set up withmembranes suitable for ammonium salts. First, a recirculating base loopcontaining about 5 wt % ammonium chloride is established. Anapproximately 3 M ammonium glycolate recirculation loop is alsoestablished. A typical batch test run is conducted at constant currentof about 0.5 to about 1.0 kA/m². The circulation loops are maintainedfor about 1 hour to about 5 hours. As the EDBM proceeds, conductivityand pH in the ammonium glycolate loop decreases. Typically, an EDBM rununder such conditions would be expected to convert at least about 80% ofthe ammonium glycolate into glycolic acid. The resulting glycolicacid/ammonium glycolate solution can be subsequently treated with astrong cationic ion exchange resin or other methods to complete theconversion.

Polymerization

The ammonium salt of a carboxylic acid comprised of a hydroxyl group canundergo condensation polymerization to form dimers, oligomers, andpolymers while liberating ammonia. The resulting polymers can beseparated from the reaction mixture using any number of techniques. Onceseparated from the reaction mixture, depolymerization can be used toobtain the free acid.

Thermal Salt Cracking of Substantially Anhydrous Ammonium Glycolate Salt

Thermal decomposition (“salt cracking”) may be used to obtain a productcomprising glycolic acid (see present Examples 62-66 and co-pending U.S.Provisional Patent Application U.S. 60/638,148; herein incorporated byreference in its entirety). This process does not require the additionof one or more chemicals prior to thermally decomposing thesubstantially anhydrous ammonium glycolate salt.

U.S. 60/638,148 describes a process to obtain glycolic acid from anaqueous solution comprising ammonium glycolate comprising:

-   -   a) providing a feed stream comprising an aqueous solution of        ammonium glycolate;    -   b) removing free water from the feed stream to produce a        substantially anhydrous salt of ammonium glycolate; and    -   c) heating the product of step b) to a temperature of less than        about 140° C. under a vacuum sufficient to remove ammonia        whereby a first liquid product mixture comprised of glycolic        acid is produced.

In one embodiment, the method may also include the steps of:

-   -   d) adding water to the first liquid product mixture of step (c)        to form a first rehydrated liquid product mixture; said        rehydrated liquid product mixture comprising glycolic acid,        glycolic acid oligomers, glycolamide, glycolic acid oligomer        ammonium salts, and unreacted ammonium glycolate; and    -   e) heating the rehydrated liquid product mixture of step (d)        under conditions whereby a portion of the glycolic acid        oligomers are hydrolyzed into free glycolic acid, wherein a        second liquid product mixture comprising glycolic acid is        formed.

Thermal salt cracking typically produces a product mixture comprisingglycolic acid. Thermal salt cracking may be combined with one or more ofthe methods described herein in order to isolate the glycolic acidproduced. In one embodiment, thermal salt cracking is used to prepare apartially deammoniated product that is subsequently used as a startingmaterial for one or more of the additional recovery methods describedherein.

The first step in the process is the removal of free water from a feedstream comprising an aqueous solution of ammonium glycolate, so that asubstantially anhydrous ammonium glycolate salt is formed (thesubstantially anhydrous salt is a viscous liquid at room temperature(˜25° C.)). Methods of removing the free water from the aqueous reactionmixture are well-known in the art including, but not limited todistillation, vacuum distillation, lyophilization, and evaporation. Inone embodiment, the free water is removed using vacuum distillation.Typically the aqueous solution of ammonium glycolate is heated to atemperature of about 40° C. to about 90° C., preferably about 40° C. toabout 80° C. under a vacuum. The vacuum pressure may vary, but istypically about 0.5 mm Hg to about 700 mm Hg absolute pressure,preferably about 0.5 mm Hg to about 635 mm Hg absolute pressure, morepreferably about 0.5 mm Hg to about 50 mm Hg absolute pressure. Thelength of time required to remove the free water may vary and can bedetermined by measuring the amount of free water removed. Typically, theamount of time required to form the substantially anhydrous salt isabout 5 minutes to about 24 hours, preferably about 5 minutes to about 8hours, more preferably about 1 hour to about 6 hours.

In one embodiment, the feed stream is heated to about 40° C. to about80° C. using a vacuum of about 5 to about 25 mm Hg absolute pressure. Inanother embodiment, the vacuum applied is about 10 mm Hg absolutepressure and the temperature is about 40° C. to about 80° C. In yetanother embodiment, the feed stream is heated to about 40° C. to about70° C.; preferably about 40° C. to about 60° C., in vacuum of about 0.5to 5 mm Hg absolute pressure; preferably at least about 1 mm Hg to about5 mm Hg absolute pressure until a substantially anhydrous salt isformed. Optionally, a non-reactive gas (e.g., nitrogen) is used to aidin the removal of water when making the anhydrous ammonium glycolatesalt. The amount of water removed can be measured using a variety oftechniques well known in the art including, but not limited to weightloss (i.e., a 25 wt % ammonium glycolate solution should lose up toabout 75% of its weight), changes in boiling temperature, and directanalysis of the of the vapor being removed. Some water may continue toevolve from the reaction mixture as side reactions (e.g., condensationpolymerization) may generate some additional water.

The next step in the process involves heating the substantiallyanhydrous salt under a vacuum to a temperature sufficient to thermallydecompose the ammonium salt into glycolic acid and ammonia as shown inEquation 5.

HOCH₂CO₂ ⁻NH₄ ⁺→NH₃+HOCH₂CO₂H  (5)

The temperature used should be chosen so that thermal decomposition ofthe salt occurs while minimizing decomposition of the acid and/orminimizing unwanted side reactions that may generate undesirablebyproducts such as glycolamide. Suitable vacuum pressures can bedetermined by one of skill in the art. An example of a typical vacuumrange is about 0.5 to about 385 mm Hg absolute pressure. In oneembodiment, the vacuum range is about 0.5 to about 80 mm Hg absolutepressure and the temperature is less than about 140° C. In anotherembodiment, the anhydrous salt is heated to a temperature of about 100°C. to about 140° C. under a vacuum of about 0.5 to about 1.5 mm Hgabsolute pressure. In yet another embodiment, the substantiallyanhydrous salt is heated to a temperature of about 120° C. to about 140°C. under a vacuum of about 0.5 to about 5 mm Hg absolute pressure. Inyet a further embodiment, the anhydrous salt is thermally decomposed ata temperature of about 120° C. to about 140° C. under a vacuum of about0.5 to about 1.5 mm Hg absolute pressure. The absolute pressure duringthe thermal decomposition of the molten ammonium salt is in partdependent on the rate of generation of ammonia gas, and may be greaterthan the absolute pressure of the vacuum applied during the thermaldecomposition. The thermal decomposition of the molten ammonium salt canuse any evaporator design, however a wipe film evaporator is preferred.

The present process includes the step of heating the substantiallyanhydrous salt of ammonium glycolate. As used herein, the term “heatingthe substantially anhydrous salt of ammonium glycolate” or “salt heatingperiod” refers to heat treatment step that includes both a time andtemperature component. The heating period used to thermally decomposethe molten ammonium glycolate salt into a fir product mixture (first“deammoniated” product mixture) comprising glycolic acid and may beadjusted depending upon the temperature and pressure used. The productsof the reaction can be monitored (i.e., ammonia generated during thermaldecomposition) to determine the amount of time necessary to obtain thedesired product. In one embodiment, the amount of ammonia released ismonitored to determine the length of time sufficient to produce thedesired deammoniated product. In another embodiment, the heating periodused to produce the deammoniated product is about 10 minutes to about 24hours, preferably about 30 minutes to about 8 hours, more preferablyabout 1 hour to about 8 hours, and most preferably about 1 hour to about6 hours.

The liquid product mixture (first liquid product mixture) produced bythermal decomposition of the anhydrous salt of ammonium glycolate isgenerally comprised of glycolic acid, oligomers of glycolic acid (bothlinear and cyclic species such as glycolide), glycolamide, ammoniumsalts of oligomers of glycolic acid, and unreacted ammonium glycolate.In one aspect, the first liquid product mixture may be further processedto chemically hydrolyze the oligomers of glycolic acid into freeglycolic acid (see Example 63). This can be accomplished by first addingwater to the liquid product mixture to produce a rehydrated liquidproduct mixture. The rehydrated liquid product mixture is subsequentlyheated for a period of time sufficient to hydrolyze at least a portionof the glycolic acid oligomers into free glycolic acid (monomeric)thereby forming a second liquid product mixture. The amount of wateradded to the first liquid product mixture may vary, but is typicallyabout 5 wt % to about 95 wt %, preferably about 20 wt % to about 80 wt%, more preferably about 40 wt % to about 60 wt %, and more preferablyabout 50 wt % based on the total weight of the resulting rehydratedliquid product mixture. As used herein, the term “heating the rehydratedliquid product mixture” is used to describe a process wherein therehydrated liquid product mixture is heated to a temperature sufficientto hydrolyze at least a portion of the glycolic acid oligomers into freeglycolic acid. In one embodiment, the heating (refluxing) conditionsincludes a temperature of about 90° C. to about 110° C., preferablyabout 100° C. to about 105° C. for a period of time from about 10minutes to about 6 hours, preferably about 30 minutes to about 6 hours,more preferably about 1 hour to about 4 hours, and most preferably about1.5 to about 3 hours.

Thermally decomposing the salt under the specified conditions converts asignificant portion of the molten ammonium glycolate salt into glycolicacid and some additional byproducts such as glycolide (cyclic dimer ofglycolic acid), linear polymeric forms of glycolic acid (typicallydimers up to pentamers), the ammonium salts of linear polymeric forms ofglycolic acid (typically dimers up to pentamers), and glycolamide. Oneof skill in the art can adjust the conditions used to thermallydecompose the ammonium glycolate to optimize free glycolic acidproduction while minimizing undesirable side reactions, such as theproduction of glycolamide. The ammonia produced during thermaldecomposition can be recovered and recycled. Optionally, the aqueousammonium glycolate solution is partially deammoniated to produce adeammoniated product that contains significantly less ammonium ion. Thisdeammoniated product is particularly attractive for subsequentprocessing as less waste (mineral salts) is generated

In addition to recovery of glycolic acid from a solution comprisingammonium glycolate, the solution comprising ammonium glycolate may berecovered directly by separation from the nitrilase catalyst by knowntechniques including but not limited to decantation or filtration, andsubsequently optionally concentrated by distillation of water from thefiltrate.

Glycolamide Byproduct Deamidation

The formation of glycolamide is an unwanted byproduct sometimesgenerated (depending upon the recovery method employed) when producingglycolic acid from ammonium glycolate. Glycolamide is formed by thereaction of ammonia with glycolic acid. Equation 6.

HOCH₂CO₂H+NH3→HOCH₂CONH₂+H₂O  (6)

However, glycolic acid can be produced from the glycolamide by reversingthis reaction (chemical hydrolysis). The can be accomplished byhydrolyzing glycolamide with water under refluxing conditions (Equation7). Optionally, the aqueous refluxing may also contain an acid or base,or an acidic or basic catalyst.

HOCH₂CONH₂+H₂0→HOCH₂CO₂H+NH₃  (7)

Alternatively, glycolamide can also react with an alcohol or polyol toliberate the corresponding ester and ammonia as shown in Equation 8.

HOCH₂CONH₂+R₂OH→HOCH₂CO₂R₂+NH₃  (8)

Alternatively, any glycolamide produced can be treated with an amidase(under appropriate conditions) to convert glycolamide to glycolic acid.Methods of converting amides to the corresponding acids using an amidase(under appropriate conditions) are known in the art. Genes encodingenzymes having amidase activity have also been cloned, sequenced, andexpressed in recombinant organisms. For example, Azza et al., (FEMSMicrobiol. Lett., 122:129 (1994)) disclose the cloning andover-expression in E. coli of an amidase gene from Brevibacterium sp.R312 under the control of the native promoter. Similarly, Kobayashi etal., (Eur. J. Biochem., 217:327 (1993)) teach the cloning of both anitrile hydratase and amidase gene from R. rhodococcus J1 and theirco-expression in E. coli. Wu et al. (DNA Cell Biol., 17:915-920 (1998);U.S. Pat. No. 6,251,650) report the cloning and overexpressing of a genefor amidase from Pseudomonas putida 5B in E. coli.

In one embodiment, the amidase activity of Comamonas testosteroni5-MGAM-4D is used to convert glycolamide to glycolic acid (ATCC 55744;U.S. Pat. No. 5,858,736, U.S. Pat. No. 5,922,589, and U.S. Ser. No.10/977,893; all hereby incorporated by reference in its entirety).Comamonas testosteroni 5-MGAM-4D has been shown to containthermally-stable, regiospecific nitrile hydratase (EC 4.2.1.84) andamidase (EC 3.5.1.4) activities useful in the conversion of a variety ofnitriles to their corresponding amides and carboxylic acids (Equation2).

Applicants specifically incorporate the entire contents of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given either as a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

General Methods

The following examples are provided to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (1994) (Phillipp Gerhardt, R. G. E.Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R.Krieg and G. Briggs Phillips, eds.), American Society for Microbiology,Washington, D.C.) or by Thomas D. Brock, in Biotechnology: A Textbook ofIndustrial Microbiology, (1989) Second Edition, (Sinauer Associates,Inc., Sunderland, Mass.).

Procedures required for genomic DNA preparation, PCR amplification, DNAmodifications by endo- and exo-nucleases for generating desired ends forcloning of DNA, ligations, and bacterial transformation are well knownin the art. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Maniatis,supra; and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, (1984) Cold Spring Harbor LaboratoryPress, Cold Spring, N.Y.; and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, (1994-1998) John Wiley & Sons, Inc., NewYork.

All reagents and materials were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma/Aldrich Chemical Company (St. Louis, Mo.)unless otherwise specified.

The abbreviations in the specification correspond to units of measure,techniques, properties, or compounds as follows: “seq” means second(s),“min” means minute(s), “h” or “hr” means hour(s), “d” means density ing/mL, “μL” means microliters, “mL” means milliliters, “L” means liters,“mM” means millimolar, “M” means molar, “mmol” means millimole(s), “wt”means weight, “wt %” means weight percent, “g” means grams, “μg” meansmicrograms, HPLC” means high performance liquid chromatography, “O.D.”means optical density at the designated wavelength, “dcw” means dry cellweight, “U” means units of nitrilase activity, “EDTA” meansethylenediaminetetraacetic acid, and “DTT” means dithiothreitol.

Analytical Methodology HPLC Analysis

The reaction product mixtures were analyzed by the following HPLCmethod. Aliquots (0.01 mL) of the reaction mixture were added to 1.50 mLof water, and analyzed by HPLC (HPX 87H column, 30 cm×7.8 mm; 0.01NH₂SO₄ mobile phase; 1.0 mL/min flow at 50° C.; 10 μL injection volume;RI detector, 20 min analysis time). The method was calibrated forglycolonitrile at a series of concentrations using commerciallyavailable glycolonitrile purchased from Aldrich.

Quantitative ¹³C NMR Analysis

Quantitative ¹³C NMR spectra were obtained using a Varian Unity Inovaspectrometer (Varian, Inc., Palo Alto, Calif.) operating at 400 MHz.Samples were prepared by taking 3.0 mL of the reaction product alongwith 0.5 mL of D₂O in a 10 mm NMR tube. ¹³C NMR spectra were typicallyacquired using a spectral width of 26 KHz with the transmitter locatedat 100 ppm, 128K points, and a 90-degree pulse (pw90=10.7 microsecondsat a transmitter power of 56 db). The longest 13C T1 (23 sec) wasassociated with the GLN nitrile carbon, and the total recycle time wasset greater than ten times this value (recycle delay d1=240 sec,acquisition time at=2.52 sec). Signal averaging of 360 scans gave atotal experiment time of 26.3 hours. The Nuclear Overhauser Enhancement(NOE) was suppressed by gating on the Waltz-modulated 1H decoupling onlyduring the acquisition time (at).

Comparative Example A Pre-Heating 0% of Formaldehyde Continuous Feed

Approximately 10.18 g of 52 wt % aqueous solution of formaldehyde (<1%methanol, E.I. DuPont de Nemours; Wilmington, Del.) was mixed with 12.81g of water, and the slurry was heated to about 76° C. for about 40 minuntil the mixture became a clear homogeneous liquid solution. Thesolution was allowed to cool to ambient temperature and remained ahomogeneous liquid. 0.14 mL of 16.7 wt % aqueous NaOH solution was thenadded to the formaldehyde solution. 1.56 g of the resulting solution (23wt % formaldehyde) was placed in the reaction vessel, and the remainderwas used for the continuous formaldehyde feed.

The reaction vessel, equipped with stirring, was placed within an oilbath maintained at 55° C. The reactants were then each continuouslypumped into the reaction vessel over a period of about 2.0 hr, asfollows:

4.41 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)7.00 mL/hr of 23 wt % aqueous formaldehyde, described above (d=1.07g/mL).

After about 2.0 hr, the feeds were stopped, the reaction vessel wasremoved from the oil bath, and the reaction mixture was quenched withthe addition of 0.07 mL of 37 wt % aqueous HCl.

FIG. 1 shows the ¹³C NMR spectrum of the resulting glycolonitrilesolution, qualitatively indicating the purity of the glycolonitrileproduct. The ¹³C NMR spectrum shows the major glycolonitrile ¹³Cresonances at about δ 48 and 119 ppm. There are also substantialresonances around δ 80-90 ppm for unreacted formaldehyde and around δ 60ppm for other by-product species derived from unreacted formaldehyde.

Example 1 Pre-Heating 90% of Formaldehyde Continuous Feed

Approximately 10.18 g of 52 wt % aqueous solution of formaldehyde (<1%methanol, DuPont) was mixed with 12.81 g of water, and the slurry washeated to about 76° C. for about 40 min until the mixture became a clearhomogeneous liquid solution. The solution was allowed to cool to ambienttemperature and remained a homogeneous liquid. 0.16 mL of 16.7 wt %aqueous NaOH solution was then added to the formaldehyde solution. 1.56g of the resulting solution (23 wt % formaldehyde) was placed in thereaction vessel, and the remainder was used for the continuousformaldehyde feed.

The reaction vessel, equipped with stirring, was placed within an oilbath maintained at 55° C. The approximately 12-inch section of theformaldehyde feed line ( 1/16″ OD (about 1.6 mm)×0.040″ ID (about 1.02mm)) directly preceding the inlet to the reaction flask was heated to120° C., and the reactants were then each continuously pumped into thereaction vessel over a period of about 2.0 hr, as follows:

4.41 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)7.00 mL/hr of 23 wt % aqueous formaldehyde, described above (d=1.07g/mL).

After about 2.0 hr, the feeds were stopped, the reaction vessel wasremoved from the oil bath, and the reaction mixture was quenched withthe addition of 0.08 mL of 37 wt % aqueous HCl.

FIG. 2 shows the ¹³C NMR spectrum of the resulting glycolonitrilesolution, qualitatively indicating the purity of the glycolonitrileproduct. Once again, the major resonances for glycolonitrile at about δ48 and 119 ppm are observed. But the resonances around δ 80-90 ppmevident in FIG. 1 for unreacted formaldehyde are noticeably reduced inFIG. 2. However, the resonances around δ 60 ppm for by-products derivedfrom unreacted formaldehyde remain, most likely due to the initialformaldehyde reactor charge.

Example 2 Pre-Heating 100% of Formaldehyde Continuous Feed

Approximately 10.18 g of 52 wt % aqueous solution of formaldehyde (<1%methanol, E.I. DuPont de Nemours) was mixed with 12.81 g of water, andthe slurry was heated to about 76° C. for about 40 min until the mixturebecame a clear homogeneous liquid solution. The solution was allowed tocool to ambient temperature and remained a homogeneous liquid. 0.14 mLof 16.7 wt % aqueous NaOH solution was then added to the formaldehydesolution. The resulting solution (23 wt % formaldehyde) was used for thecontinuous formaldehyde feed.

The reaction vessel, equipped with stirring, was charged with a mixtureof 0.18 g of HCN in 3.4 g of water and then placed within an oil bathmaintained at 55° C. The approximately 12-inch section of theformaldehyde feed line ( 1/16″ OD×0.040″ ID) directly preceding theinlet to the reaction flask was heated to 120° C., and the reactantswere then each continuously pumped into the reaction vessel over aperiod of about 2.0 hr, as follows:

4.41 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)7.67 mL/hr of 23 wt % aqueous formaldehyde, described above (d=1.07g/mL).

After about 2.0 hr, the feeds were stopped, the reaction vessel wasremoved from the oil bath, and the reaction mixture was quenched withthe addition of 0.07 mL of 37 wt % aqueous HCl.

FIG. 3 shows the ¹³C NMR spectrum of the resulting glycolonitrilesolution, qualitatively indicating the purity of the glycolonitrileproduct. The major resonances for glycolonitrile at about δ 48 and 119ppm are evident in FIG. 3, while the levels of impurities aresubstantially reduced from the levels observed in FIG. 1 and FIG. 2.

Example 3 Pre-Heating 100% of Formaldehyde Continuous Feed

Approximately 14.20 g of 37 wt % aqueous solution of formaldehyde(10-15% methanol, Acros Organics, Morris Plains, N.J.) was mixed with8.78 g of water and 0.14 mL of 16.7 wt % aqueous NaOH. The resultingsolution (23 wt % formaldehyde) was used for the continuous formaldehydefeed.

The reaction vessel, equipped with stirring, was charged with a mixtureof 0.18 g of HCN in 3.4 g of water and then placed within an oil bathmaintained at 55° C. The approximately 12-inch section of theformaldehyde feed line ( 1/16″ OD×0.040″ ID) directly preceding theinlet to the reaction flask was heated to 120° C., and the reactantswere then each continuously pumped into the reaction vessel over aperiod of about 2.0 hr, as follows:

4.21 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)7.67 mL/hr of 23 wt % aqueous formaldehyde, described above (d=1.07g/mL).

After about 2.0 hr, the feeds were stopped, the reaction vessel wasremoved from the oil bath, and the reaction mixture was quenched withthe addition of 0.07 mL of 37 wt % aqueous HCl.

FIG. 4 shows the ¹³C NMR spectrum of the resulting glycolonitrilesolution, qualitatively indicating the purity of the glycolonitrileproduct. Again, the major resonances for glycolonitrile at about δ 48and 119 ppm are evident in FIG. 4, while the levels of impurities aresubstantially reduced from the levels observed in FIG. 1 and FIG. 2.FIG. 4 also clearly shows the resonance at δ 49 ppm for the methanolfrom the formalin feed used in Example 3.

Examples 4-8 Pre-Heating 100% of Formaldehyde Continuous Feed

In Examples 4-8, the following glycolonitrile synthesis procedure wasrepeated five separate times.

Approximately 0.56 mL of 16.7 wt % aqueous NaOH solution was added to218.0 g of 37 wt % aqueous solution of formaldehyde (containing 7 wt %to 8 wt % methanol). The resulting solution was used for the continuousformaldehyde feed.

The reaction vessel, equipped with a magnetic stirbar, was initiallycharged with a mixture of 3.3 g HCN in 35.3 g water and placed within awater bath maintained at around 20° C., on top of a stirplate and labjack assembly in a lowered position. The approximately 36-inch sectionof the formaldehyde feed line (⅛″ OD (about 3.18 mm)×0.085″ ID (about2.16 mm)) directly preceding the inlet to the reaction flask was heatedto 120° C. after filling the formaldehyde feed line, and the flow ofheated formaldehyde feed was first established by observing two-phaseflow from the outlet of the formaldehyde feed line. After establishingtwo-phase flow out of the formaldehyde feed line, the reaction vesselwas raised to introduce the formaldehyde feed directly into the liquidreaction mixture. The stirplate, water bath, and lab jack assembly wasthen raised accordingly to provide reactor mixing and to maintain thereaction temperature around 20-25° C., which was accomplished byperiodically adding ice and/or dry ice to the external water bath.

The reactants were each continuously pumped into the reaction vesselover a period of about 2.0 hr, as follows:

82.4 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)92.7 mL/hr of 37 wt % aqueous formaldehyde, described above (d=1.09g/mL).

After 2.0 hr, the feeds were stopped, and the reaction vessel, waterbath, stirplate, and lab jack assembly was lowered to remove theformaldehyde feed line from the reaction product. The reaction mixturewas removed from the reaction vessel and then quenched by adding of 1.3mL of an aqueous solution of 70% glycolic acid (70% Glypure®; E.I.DuPont de Nemours, Wilmington, Del.), resulting in a glycolonitrileproduct solution at about pH 3.

Each of the glycolonitrile reaction product solutions was individuallyconcentrated to remove the excess unreacted HCN and the methanol fromthe commercial source of formaldehyde. The concentration step wasperformed under vacuum with mild heating using an external oil bath at60-70° C.

The weight of each concentrated glycolonitrile product solution wasrecorded, and the glycolonitrile concentration was determined by HPLC.

The conditions used in Examples 4-8, and the resulting GLN yield isreported in Table 1.

TABLE 1 Glycolonitrile Yield. Weight of GLN Glycolonitrile Yield Example# Solution (g) Concentration (M) (% recovered GLN) 4 116 13.9 61% 5 13917.7 94% 6 163 13.7 85% 7 150 16.7 95% 8 182 11.6 80%

The five concentrated glycolonitrile product solutions produced inExamples 4-8 were combined into a composite product sample, and aquantitative ¹³C NMR analysis was performed on the composite sample todetermine the purity of the glycolonitrile produced. FIG. 5 shows the¹³C NMR spectrum of the composite sample. The quantitative ¹³C NMRanalysis showed that the glycolonitrile product purity was greater than99.9% in the composite sample.

Example 9 Pre-Heating 100% of Formaldehyde Continuous Feed

Approximately 0.27 mL of 16.7 wt % aqueous NaOH solution was added to54.5 g of 37 wt % aqueous solution of formaldehyde (containing 7-8%methanol). The resulting solution was used for the continuousformaldehyde feed.

The reaction vessel, equipped with a magnetic stirbar, was initiallycharged with a mixture of 0.29 g HCN in 10.3 g water and placed within awater bath maintained at around 25° C., on top of a stirplate. Theapproximately 12-inch section of the formaldehyde feed line (⅛″OD×0.085″ ID) directly preceding the inlet to the reaction flask washeated to 150° C. after filling the formaldehyde feed line, and the flowof heated formaldehyde feed was first established outside the reactionvessel by observing two-phase flow from the outlet of the formaldehydefeed line. After establishing heated formaldehyde feed, the end of theformaldehyde feed line was placed directly into the liquid reactionmixture. The reaction temperature was maintained around 20-25° C., whichwas accomplished by periodically adding ice and/or dry ice to theexternal water bath. The reactants were each continuously pumped intothe reaction vessel over a period of about 2.0 hr, as follows:

7.02 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)7.67 mL/hr of 37 wt % aqueous formaldehyde, described above (d=1.09g/mL).

After 2.0 hr, the feeds were stopped, and the formaldehyde feed line wasremoved from the reaction product. The reaction mixture was removed fromthe reaction vessel and then quenched by adding of 0.060 mL of 70%Glypure® glycolic acid, resulting in a glycolonitrile product solutionat about pH 3.

FIG. 6 shows the ¹³C NMR spectrum of the resulting glycolonitrilesolution, qualitatively indicating the purity of the glycolonitrileproduct.

Example 10 Pre-Heating 100% of Formaldehyde Continuous Feed

Approximately 0.40 mL of 16.7 wt % aqueous NaOH solution was added to58.0 g of 37 wt % aqueous solution of formaldehyde (containing 7-8%methanol).

The resulting solution was used for the continuous formaldehyde feed.

The reaction vessel, equipped with a magnetic stirbar, was initiallycharged with a mixture of 0.29 g HCN in 10.3 g water and placed within awater bath maintained at around 25° C., on top of a stirplate. Theapproximately 24-inch section of the formaldehyde feed line (⅛″OD×0.085″ ID) directly preceding the inlet to the reaction flask washeated to 90° C. after filling the formaldehyde feed line. Afterestablishing heated formaldehyde feed outside of the reaction vessel,the end of the formaldehyde feed line was placed directly into theliquid reaction mixture. The reaction temperature was maintained around20-25° C., which was accomplished by periodically adding ice and/or dryice to the external water bath. The reactants were each continuouslypumped into the reaction vessel over a period of about 2.0 hr, asfollows:

7.02 mL/hr of 50 wt % aqueous HCN solution (d=0.86 g/mL)7.67 mL/hr of 37 wt % aqueous formaldehyde, described above (d=1.09g/mL).

After 2.0 hr, the feeds were stopped, and the formaldehyde feed line wasremoved from the reaction product. The reaction mixture was removed fromthe reaction vessel and then quenched by adding of 0.10 mL of 70%Glypure® glycolic acid, resulting in a glycolonitrile product solutionat about pH 3-4.

FIG. 7 shows the ¹³C NMR spectrum of the resulting glycolonitrilesolution, qualitatively indicating the purity of the glycolonitrileproduct.

Example 11 Construction of High Copy Nitrilase Expression Plasmid

Synthetic Oligonucleotide Primers

165 (5′-CGACTGCAGTAAGGAGGAATAGGACATGGTTTCGTATAACAGC AAGTTC-3′; SEQ IDNO: 1) and 166 (5′-TGATCTAGAGCTTGGAGAATAAAGGGGAAGACCAGAGAT G-3′; SEQ IDNO: 2)(which incorporate PstI and XbaI restriction sites (underlined),respectively) were used to PCR amplify the nitrilase gene from A.facilis 72W (ATCC 55746) genomic DNA (SEQ ID NO:5).

Typical PCR parameters are as follows:

Step 1: 5 minutes at 95° C.Step 2: 0.5 minute at 95° C. (denaturation)Step 3: 0.5 minute at 55° C. (annealing)Step 4: 1 minute at 74° C. (extension)Steps 2-4 are repeated 25 cycles

PCR reagents are supplied by and used as recommended by RocheDiagnostics Corporation (Indianapolis, Ind.).

The only change from native Acidovorax facilis 72W sequence is a changeto the first nucleotide from G to A to facilitate expression in E. coli.In so doing, the start codon of the nitrilase gene was changed from thenative GTG to ATG. Accordingly, the first amino acid of thecorresponding nitrilase protein is changed from the native valine tomethionine (SEQ ID NO: 6). Oligonucleotide primer 165 also introduces aribosome binding site (bold) and a codon (TAG) to stop translation oflacZ prior to initiation of translation of nitrilase. The PCR productwas digested with PstI and XbaI, and cloned into pUC19 (GenBank® L09137;New England Biolabs, Beverly, Mass.) digested with PstI and XbaI, togenerate the plasmid identified as pSW138.

Example 12 Expression of Active Nitrilase in E. coli

Plasmid pSW138 was used to transform E. coli MG1655 (ATCC 47076) and E.coli FM5 (ATCC 53911) to generate the two strains identified as (1)MG1655/pSW138 and (2) FM5/pSW138, respectively. Each strain was grown,induced, harvested, and assayed for nitrilase activity (conversion ofglycolonitrile to glycolic acid) as described below. Replicates of sixare performed for each strain.

1. Bacterial Growth

Strain inoculums were grown in LB media supplemented with ampicillin (50mg/L), at 37° C. with shaking (200 rpm) for 16-18 hours.

2. Induction of Nitrilase Expression

Sufficient inoculum was added to fresh LB media supplemented withampicillin (50 mg/L) and IPTG (1 mM) to give an initial OD (600 nm) ofapproximately 0.1. Cultures were incubated at 37° C. with shaking (200rpm) for approximately 6-8 hours.

3. Bacterial Harvest

Bacterial cells were harvested by centrifugation, removing as muchliquid as possible, and cell pellets were frozen at −70° C.

4. Assay for Nitrilase Activity

Into a temperature controlled (25° C.) 20-mL glass scintillation vialequipped with a micro stir bar was added 3.0 mL of substrate solution(0.667 M glycolonitrile; TCl) and 1.0 mL of cell suspension (400 mg wetcell weight/mL in 100 mM sodium pyrophosphate pH 6.0, 1 μg/mL DNAse).Final glycolonitrile concentration was 500 mM and final cellconcentration was 100 mg/mL. Samples (100 μL) were removed at 5, 10, 15,30, 45 and 60 min and added to assay mix (100 μL deionized water, 3 μL6.0 N HCl, 200 μL 200 mM n-propanol), followed by vortexing andcentrifugation. Resulting supernatants were analyzed by HPLC (HPX 87Hcolumn, 30 cm×7.8 mm; 0.01 NH₂SO₄ mobile phase; 1.0 mL/min flow at 50°C.; 10-μL injection volume; 20 min analysis time) for glycolonitrile(GLN) and glycolic acid (GLA) Dry cell weights (dcw) were determined onduplicate samples by microwave drying. Nitrilase activity was reportedas U/g dcw, where 1 unit (U) converts 1 μmol of GLN to GLA in 1 min at25° C. (Table 2).

TABLE 2 Strain Nitrilase activity (U/g dcw) MG1655/pSW138 22.1FM5/pSW138 3.3

Example 13 Construction of A. facilis 72W Nitrilase Random MutagenesisLibraries by Error-Prone Polymerase Chain Reaction

Genomic DNA was prepared from A. facilis 72W (ATCC 55746) using aPuregene® DNA isolation kit according to the manufacturer's instructions(Gentra Systems, Minneapolis, Minn.). Error-prone PCR was performed onthe A. facilis 72W nitrilase gene (coding sequence; SEQ ID NO:5) usingprimers identified as SEQ ID NO: 3 (5′-GCGCATATGGTTTCGTATAACAGCAAGTTCC-3′) and SEQ ID NO: 4(5′-ATAGGATCCTTATGGCTACTTTGCTGGGACCG-3′) according to instructionssupplied with the GeneMorph® PCR Mutagenesis Kit (Stratagene, La Jolla,Calif.). Reaction conditions recommended to produce a low mutationfrequency (0-3 mutations/kb) and a medium mutation frequency (3-7mutations/kb) were employed. Ten percent of the 1.1 kb PCR product wasligated into the expression vector pTrcHis2 TOPO® according toinstructions supplied with the pTrcHis2 TOPO® TA Expression kit(Invitrogen, Carlsbad, Calif.). One half of the ligation mixture wastransformed into E. coli TOP10 according to supplier's recommendations(Invitrogen). One percent of the transformation mixture was plated ontoLB plates supplemented with 50 mg/L ampicillin. Resultant transformantsnumbered 200-400 colonies, suggesting that the total PCR productproduced was capable of generating 400,000-800,000 colonies, more thanenough required to screen for improved enzyme activity. Mutationfrequencies were confirmed by nucleotide sequence analysis of a randomlyselected sample of clones. Sequence analysis also confirmed thatapproximately 50% of inserts were in the forward orientation, asexpected. SDS-PAGE analysis confirmed that essentially all clones withforward orientation inserts expressed the ˜41 kDa nitrilase protein whengrown and induced as recommended (Invitrogen).

In addition, the native A. facilis 72W nitrilase gene was amplified bystandard PCR using primers identified as SEQ ID NO: 3 and SEQ ID NO: 4,and the resulting DNA product was cloned into pTrcHis2-TOPO®(Invitrogen) according to manufacturer recommendations, to generate theplasmid pNM18. Transformation of E. coli TOP10 or E. coli FM5 (ATCC53911) with pNM18 produced strains useful as respective controls. The A.facilis 72W nitrilase “control” sequence in pNM18 (SEQ ID NO: 5) isidentical to the coding sequence of the wild-type A. facilis 72W exceptfor a change in the start codon from GTG to ATG, facilitating expressionin E. coli.

Example 14 Screening A. facilis 72W Nitrilase Random MutagenesisLibraries for Increased Nitrilase Activity

Approximately 10,000 colonies from the low mutation frequencyerror-prone PCR library (constructed as described in Example 13) wereplated on LB agar supplemented with 50 mg/L ampicillin. High throughputscreening was performed in 96-well microtiter plates using robotics.After growth of individual colonies in liquid LB supplemented with 50mg/L ampicillin and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for18 h at 37° C., 200 rpm shaking, cultures were supplied with 50 mMglycolonitrile (GLN) for 1 h at 37° C., 80 Hz linear shaking. Reactionswere stopped by filtering out the bacterial cells, and supernatants tobe analyzed were sealed in microtiter plates and stored at 4° C. untilanalysis.

Production of glycolic acid (GLA) was measured by atmospheric pressurechemical ionization (APCI) mass spectrometry in the negative ion modemonitoring the M-H ion, m/z 75, in single ion mode. The massspectrometer used was a Micromass (Waters) Quattro Ultima triple quad,with the following settings: source temperature=150° C., probetemperature=300° C., cone gas=80 L/hr, Desolvation gas=700-800 L/hr.Cone voltage=35 V, Corona voltage=20 mA. Multiplier=600 V, Dwell=0.1 s,Interchannel Delay=0.02 s. The mobile phase was 50/50 MeOH/H₂O at 3.5mL/min per needle with a 1:5 split of eluent before introduction intothe mass spectrometer using a LC Packings Acurate splitter. Samples weredelivered by a Gilson 215 auto-sampler with a 889 serial injection8-valve bank injecting 30 mL of sample into 5-mL sample loops. A HudsonPlate Crane XT plate handling robot delivered plates to the deck of theGilson auto-sampler. A needle and injection port wash with the samesolvent at 5 mL/min was performed between each set of 8 injections. Bythis method, seven strains with increased nitrilase activity wereidentified and isolated.

Example 15 Identification of Mutations in A. facilis 72W NitrilaseConferring Increased Nitrilase Activity

Nucleotide sequence analysis was used to identify any mutations presentin the nitrilase gene of the seven TOP10 mutant strains isolated asdescribed in Example 14, and the corresponding amino acid changes werededuced. All seven strains showed the identical nitrilase sequence (SEQID NO: 8), with a single amino acid change, Leu at position 201 changedto Gln (L201Q) in the plasmid identified as pNM18-201Q. This change hadno detectable effect on nitrilase protein production (compared to thenative enzyme), as measured by SDS-PAGE analysis.

Example 16 Saturation Mutagenesis of Nitrilase at Amino Acid ResiduePosition 201

A saturation mutagenesis library at amino acid position 201 of the A.facilis 72W nitrilase enzyme was constructed using degenerateoligonucleotides and the QuikChange® Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions. Approximately 500 members of this library were screenedfor increased nitrilase activity as previously described (Example 14).Nucleotide sequencing analysis was used to determine any amino acidchanges at position 201 that conferred increased nitrilase activity. Inaddition to L201Q (SEQ ID NO: 8), the following mutations conferringincreased nitrilase activity were identified from the screen: L201 G(SEQ ID NO: 16), L201H (SEQ ID NO: 18), L201K (SEQ ID NO: 20), L201N(SEQ ID NO: 22), L201S (SEQ ID NO: 24), L201A (SEQ ID NO:10), L201C (SEQID NO: 12), and L201T (SEQ ID NO: 14) in the plasmids identified aspNM18-201G, pNM18-201H, pNM18-201K, pNM18-201N, pNM18-201S, pNM18-201A,pNM18-201C, and pNM18-201T, respectively.

Example 17 Targeted Saturation Mutagenesis of the A. facilis 72WNitrilase Catalytic Domain

We hypothesized that the catalytic domain within the A. facilis 72Wnitrilase (SEQ ID NO: 6) may be a suitable region to mutate in anattempt to increase nitrilase activity toward 2-hydroxynitriles, namelyglycolic acid.

Saturation mutagenesis within the A. facilis 72W nitrilase (SEQ ID NO:6) catalytic domain (160G 161G 162L 163N 164C 165W 166E 167H 168F 169Q170P 171L 172S 173K) of those residues not universally conserved amongknown bacterial nitrilases (underlined) was completed using degenerateoligonucleotides and the QuikChange® Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.) according to the manufacturer'sinstructions. Specifically, nine mini-libraries (500-1000 colonies) wereconstructed, one for each of the active site residues targeted(underlined above). These libraries were screened for increasednitrilase activity as previously described. Nucleotide sequencinganalysis was used to determine any amino acid changes that conferredincreased nitrilase activity. The following changes conferring increasednitrilase activity were identified: F168K (SEQ ID NO: 26), F168M (SEQ IDNO: 28), F168T (SEQ ID NO: 30), and F168V (SEQ ID NO: 32) in theplasmids identified as pNM18-168K, pNM18-168M, pNM18-168T, andpNM18-168V, respectively.

Example 18 Construction of MG1655/ISW138-168K, MG1655/ISW138-168M,MG1655/ISW138-168T, MG1655/ISW138-168V, MG1655/ISW138-201Q,MG1655/ISW138-201G, MG1655/ISW138-201H, MG1655/ISW138-201K,MG1655/ISW138-201N, and MG1655/ISW138-201S

Each of the plasmids pNM18-168K, pNM18-168M, pNM18-168T, pNM18-168V,pNM18-201Q, pNM18-201G, pNM18-201H, pNM18-201K, pNM18-201N, andpNM18-201S was cleaved with EcoRI and the smaller EcoRI fragment (907bp) was subcloned into the plasmid pSW138 (described in Example 11)which had also been cleaved with EcoRI, to generate the plasmidspSW138-168K, pSW138-168M, pSW138-168T, pSW138-168V, pSW138-201Q,pSW138-201G, pSW138-201H, pSW138-201K, pSW138-201N, and pSW138-201S,respectively. Each of the plasmids pSW138-168K, pSW138-168M,pSW138-168T, pSW138-168V, pSW138-201Q, pSW138-201G, pSW138-201H,pSW138-201K, pSW138-201N, and pSW138-201S was used to transform E. coliMG1655 to generate the strains MG1655/pSW138-168K, MG1655/pSW138-168M,MG1655/pSW138-168T, MG1655/pSW138-168V, MG1655/pSW138-201Q,MG1655/pSW138-201G, MG1655/pSW138-201H, MG1655/pSW138-201K,MG1655/pSW138-201N, and MG1655/pSW138-201S; respectively.

Example 19 Nitrilase Activity of Mutants Produced by 10-LiterFermentation

E. coli seed cultures were grown in 500 mL LB media supplemented with0.1 mg ampicillin per mL for 6-10 h (OD₅₅₀=1-2) at 30° C. with shaking(300 rpm) prior to inoculation of the fermentor.

Growth of nitrilase strains was in 14-L Braun Biostat C fermentors (B.Braun Biotech International Gmbh, Melsungen, Germany) using mineralmedium with glucose, ammonia, and salts. IPTG (for FM5/pNM18 basedstrains) or lactose (for MG1655/pSW138 based strains) was used forinduction.

Pre-sterilization fermentor media (7.5 L) is described in Table 3.Post-sterilization additions include filter sterilized trace elements(Table 4), 0.1 mg ampicillin per mL, 2 g casamino acids (Difco) per L, 4g glucose per L, and 500 mL seed culture.

Fermentation set points are described in Table 5. NH₄OH (40% w/v) andH₂PO₄ (20% w/v) were used for pH control. The dissolved oxygenconcentration was controlled at 25% of air saturation with the agitationto rise first with increase oxygen demand and the aeration to follow.The fermentation feed protocols used with IPTG induction and lactoseinduction are given in Tables 6 and 7, respectively. Glucose feed rateswere reduced if glucose accumulated above 5 g/L. For FM5/pNM18 basedstrains, IPTG was added to 0.5 mM at OD₅₅₀=20-30. After 40-56 hrs cellswere chilled to 5-10° C. and harvested by centrifugation. Nitrilaseactivity was determined as described (Example 12) and results are shownin Table 8.

TABLE 3 Fermentation media, pre-sterilization. (NH₄)₂SO₄ 5.0 g/L K₂HPO₄4.0 g/L KH₂PO₄ 3.5 g/L MgSO₄*7H₂O 0.6 g/L Na₃Citrate*2H₂O 1.0 g/L NZAmine AS (Quest) 2.5 g/L Antifoam - Biospumex 153K 0.25 mL/L

TABLE 4 Fermentation trace elements Concentration Citric acid 10 g/LCaCl₂*2H₂O 1.5 g/L FeSO₄*7H₂O 5 g/L ZnSO₄*7H₂O 0.39 g/L CuSO₄*5H₂O 0.38g/L CoCl₂*6H₂O 0.2 g/L MnCl₂*4H₂O 0.3 g/L

TABLE 5 Fermentation set points Initial Set-Point Minimum MaximumStirrer (rpm) 400 400 1000 Airflow (slpm) 2 2 10 pH 6.8 6.8 6.8 Pressure(kPa) 0.5 0.5 0.5 DO 25% 25% 25% Temperature 30 30 30 ° C.

TABLE 6 Fermentation feed protocol used with IPTG induction Feed RateEFT (hr) (g/min) Substrate 0 0 Glucose (batched) 5 0.27 Glucose (50%w/w)

TABLE 7 Fermentation feed protocol used with lactose induction Feed RateEFT (hr) (g/min) Substrate 0 0 Glucose (batched) 5 0.27 Glucose (50%w/w) 14 1.3 Lactose (25% w/w)

TABLE 8 Nitrilase Activity for Mutants Grown in 10-Liter FermentationsNitrilase Fold Activity Increase vs. Mutation (GLA Respective (SEQ IDNO.) E. coli Strain U/g dcw) Control None FM5/pNM18 (control) 387 NA(SEQ ID NO: 6) None MG1655/pSW138 (control) 490 NA (SEQ ID NO: 6) F168KFM5/pNM18-168K 1250 3.2 (SEQ ID NO: 26) F168K MG1655/pSW138-168K 12302.5 (SEQ ID NO: 26) F168M MG1655/pSW138-168M 1261 2.6 (SEQ ID NO: 28)F168T FM5/pNM18-168T 2152 5.6 (SEQ ID NO: 30) F168T MG1655/pSW138-168T837 1.7 (SEQ ID NO: 30) F168V MG1655/pSW138-168V 1763 3.6 (SEQ ID NO:32) L201Q FM5/pNM18-201Q 2603 6.7 (SEQ ID NO: 8) L201QMG1655/pSW138-201Q 2410 4.9 (SEQ ID NO: 8) L201G FM5/pNM18-201G 2985 7.7(SEQ ID NO: 16) L201H FM5/pNM18-201H 2322 6.0 (SEQ ID NO: 18) L201HMG1655/pSW138-201H 1334 2.7 (SEQ ID NO: 18) L201K FM5/pNM18-201K 443411.5 (SEQ ID NO: 20) L201N FM5/pNM18-201N 2542 6.6 (SEQ ID NO: 22) L201NMG1655/pSW138-201N 2695 5.5 (SEQ ID NO: 22) L201S FM5/pNM18-201S 14633.8 (SEQ ID NO: 24)

Example 20 Determination of Nitrilase Activity for E. coli TOP10/pNM18,E. coli TOP10/pNM18-201A, E. coli TOP10/pNM18-201C, and E. coliTOP10/pNM128-201T (Shake Flask)

In duplicate, 10 mL of an overnight culture (LB+50 μg/mL ampicillin, 37°C. with shaking) was added to 200 mL (LB+50 ug/ml ampicillin+1 mM IPTG)and incubated at 37° C. with shaking for 4-5 hrs (final OD600approximately 2.0). Cells were collected by centrifugation at 4° C. andstored frozen at −80° C.

To a 4-mL glass vial equipped with a magnetic stir bar was added 1.0 mLof 1.0 M glycolonitrile in water, and the vial and its contentsequilibrated to 25° C. in a temperature controlled water bath. Withstirring, 1.0 mL of 0.100 M potassium phosphate buffer (pH 7.0)containing 40-100 mg wet cell paste pre-equilibrated to 25° C. was addedto the vial (final [GLN]=0.5M). Samples (0.100 mL) were taken atpredetermined times and mixed with a solution comprised of 0.100 mLwater, 0.020 mL of 6.0 N acetic acid and 0.200 mL of 0.20 M sodiumbutyrate in water (HPLC external standard). The resulting mixture wascentrifuged and the resulting supernatant analyzed by HPLC for glycolicacid using a Supelco® (Sigma Aldrich Corp.) LC-18-DB column (15 cm×4.6mm): mobile phase: aqueous 10 mM sodium acetate (NaOAc), 10 mM aceticacid (AcOH), 7.5% (v/v) methanol. The dry cell weight (dcw) of each cellpaste was determined and used to calculate cell-specific nitrilaseactivity. Table 9 summarizes the increases in nitrilase activity for thenitrilase mutants compared to the native nitrilase.

TABLE 9 Nitrilase activity for mutants L201A, L201C, and L201T versuscontrol (shake flasks). Nitrilase Activity Fold Increase Mutation (GLAin activity (SEQ ID NO.) E. coli strain U/g dcw) (vs. control) NoneTOP10/pNM18 135 NA (SEQ ID NO: 6) (Control) L201A TOP10/pNM18-201A 3712.7 (SEQ ID NO: 10) L201C TOP10/pNM18-201C 289 2.1 (SEQ ID NO: 12) L201TTOP10/pNM18-201T 308 2.3 (SEQ ID NO: 14)

Example 21 Preparation of Immobilized E. coli SS1001 (ATCC PTA-1177)

E. coli strain SS1001 (ATCC PTA-1177) is a transformed E. coli strainexpressing the Acidovorax facilis 72W nitrilase (U.S. Pat. No.6,870,038; herein incorporated by reference). The coding sequence of therecombinantly expressed (E. coli SS1001) nitrilase (SEQ ID NOs: 37-38)contains 2 minor sequence changes in comparison to the wild-type 72Wnitrilase sequence (SEQ ID NO: 5). The start codon was changed from GTGto ATG to facilitate recombinant expression and an artifact wasintroduced during cloning that resulted in a single amino acid changenear the C-terminal (Pro367 [CCA]→Ser [TCA]).

This strain was grown in a 10-L fermentation as previously described(see Example 8 of U.S. Ser. No. 10/919,182), and the cell paste(glycolonitrile (GLN) was used in a process to convert GLN to glycolicacid (GLA) as follows.

E. coli SS1001 cells were first immobilized in carrageenan beads (theimmobilized E. coli SS1001) according to the following procedure. Withrapid stirring, 9 g of carrageenan (FMC GP911; FMC Corp., Philadelphia,Pa.) was slowly added to 231 g deionized distilled water at 50° C., theresulting mixture heated to 80° C. until carrageenan was completelydissolved, and the resulting solution cooled with stirring to 47° C. Ina separate beaker equipped with stir bar, 75.9 g of frozen E. coliSS1001 cells (39.53% dcw) was added to 84.1 g of 0.35 M Na₂HPO₄ (pH 7.3)at ca. 25° C. and mixed until the cells were suspended, then adeoxyribonuclease I solution (10 μL of 12,500 U/mL DNase (Sigma Aldrich,St. Louis, Mo.)/100 mL of cell suspension) was added. The cellsuspension was heated with stirring to 45-46° C. immediately beforeaddition to carrageenan solution. With stirring, 160.0 g of E. coliSS1001 cell suspension at 47° C. was added to the carrageenan solutionat 47° C., and the resulting cell/carrageenan suspension was pumpedthrough an electrically-heated 20 gauge needle at 47° C. and drippedinto 0.25 M KHCO₃ (pH=7.3) with stirring at room temperature (ca. 21-22°C.); the flow rate through the needle was set at 5-8 mL/min. Theresulting beads were allowed to harden for 1 h with stirring, and werestored in 0.25 M KHCO₃ (pH 7.3). Chemical crosslinking of the beads wasperformed by addition of 0.5 g of 25% glutaraldehyde (GA) in water(Sigma M 752-07) to 20 g beads suspended in 48 mL of 0.25 M KHCO₃ (pH7.3), and stirring for 1 h at room temperature. To the suspension ofbeads was then added 2.0 g of 12.5 wt % polyethylenimine (PEI, BASFLUPASOL PS; BASF Aktiengesellschaft, Ludwigshafen, Germany) in waterfollowed by mixing for an additional 1 h at room temperature. TheGA/PEI-crosslinked beads were stored in 1.0 M NH₄HCO₃ (pH 7.3) at 5° C.

Biocatalytic conversion of GLN to GLA was followed by HPLC. Aliquots(0.2 mL) of the reaction mixture were added to 0.01 mL 6 M HCl and 0.8mL of 0.25 M n-propanol in water (HPLC external standard), and analyzedby HPLC (HPX 87H column (Bio-Rad, Hercules, Calif.), 30 cm×7.8 mm; 0.01N H₂SO₄ mobile phase; 1.0 mL/min flow at 50° C.; 10 μL injection volume;refractive index (RI) detector, 20 min analysis time) for GLN and GLA.The nitrilase activity of the GA/PEI-crosslinked carrageenan/7.5% (dcw)E. coli SS1001 beads, was ˜12 U/g beads, where 1 unit (U) converts 1μmol of GLN to GLA in 1 min at 25° C.

Example 22 Conversion of 1 M Glycolonitrile (GLN) to Ammonium Glycolate(NH₄GLA)

A 50-mL jacketed reaction vessel with overhead stirring was charged with4 g E. coli SS1001 beads (Example 21), 13.73 mL deionized water, 0.4 mL5 M NH₄GLA, and 1.87 mL GLN (ca. 52 wt % in water (TCl)), 0.89 M GLNfinal concentration, pH adjusted to pH 7.6, the mixture stirred at 25°C., and 0.2 mL aliquots were taken out to follow the reaction progressby HPLC. When all GLN was converted to NH₄GLA, product solution wasdecanted, 14.13 mL deionized water and 1.87 mL GLN were added tobiocatalyst, pH adjusted to pH 7.6, and biocatalyst recycle wasrepeated. The initial rate of NH₄GLA synthesis at the first biocatalystrecycle was 143 mM/h. Percent decrease in initial rate of NH₄GLAsynthesis vs. the recycle number are shown in Table 10 (“1 M”).

Example 23 Conversion of Approximately 3 M Glycolonitrile (GLN) toAmmonium Glycolate (NH₄GLA)

A 50-mL jacketed reaction vessel with overhead stirring was charged with4 g E. coli SS1001 beads (Example 11), 6.39 mL deionized water, 4 mL 1 MKHCO₃, and 5.61 mL GLN (ca. 52 wt % in water (TCl)), 2.68 M GLN finalconcentration, pH was adjusted to pH 7.6, the mixture stirred at pH 25°C., and 0.2 mL aliquots were taken out to follow the reaction progressby HPLC. When all GLN was converted to NH₄GLA, product solution wasdecanted, 6.39 mL deionized water, 4 mL 1 M KHCO₃, and 5.61 mL GLN wereadded to biocatalyst, pH adjusted to pH 7.6, and biocatalyst recycle wasrepeated. The initial rate of NH₄GLA synthesis at the first biocatalystrecycle was 207 mM/h. Percent decrease in initial rate of NH₄GLAsynthesis vs. the recycle number are shown in Table 10 (“3 M”).

Example 24 Addition of Approximately 3 M Glycolonitrile in Approximately1 M Increments (1 M+1 M+1 M) to Yield Ammonium Glycolate (NH₄GLA)

A 50-mL jacketed reaction vessel with overhead stirring was charged with4 g E. coli SS1001 beads (Example 21), 8.13 mL deionized water, 4 mL 1 MKHCO₃, and 1.87 mL GLN (ca. 52 wt % in water (TCl)), 0.89 M GLN, pH wasadjusted to pH 7.6, the mixture stirred at 25° C., and 0.2 mL aliquotswere taken out to follow the reaction progress by HPLC. When all GLN wasconverted to NH₄GLA, second portion of 1.87 mL GLN was added, pH wasadjusted to pH 7.6, and when all GLN was consumed, the third portion of1.87 mL GLN was added, pH adjusted to pH 7.6, and reaction completedyielding approximately 3 M NH₄GLA solution. Product solution wasdecanted, 8.13 mL deionized water, 4 mL 1 M KHCO₃, and 1.87 mL GLN wereadded to biocatalyst, pH adjusted to pH 7.6, GLN conversion proceeded tocompletion, and addition of GLN, water and buffer, pH adjustment, andcompletion of GLN conversion were repeated twice more to finish therecycle (step-wise conversion of GLN in three approximately 1 Mincrements), and biocatalyst recycles were repeated. The initial rate ofNH₄GLA synthesis in the first 1 M GLN solution in a first recycle (three˜1 M portions of GLN per recycle) was 155 mM/h. Percent decrease ininitial rate of NH₄GLA synthesis vs. the recycle number are shown inTable 10 (“(1 M+1 M+1 M)=3 M”).

Example 25 Continuous Addition of Glycolonitrile (GLN) to 0.2 M GLN toYield Ammonium Glycolate (NH₄GLA)

A 50-mL jacketed reaction vessel with overhead stirring was charged with4 g E. coli SS1001 beads (Example 21), 8 mL deionized water, 4 mL 1 MKHCO₃, and 0.4 mL GLN (ca. 52 wt % in water (TCl)), pH was adjusted topH 7.6, the mixture stirred at 25° C., GLN solution was continuouslyadded up to 3 M GLN at a rate of GLN consumption to keep GLNconcentration around 0.2 M, and 0.2 mL aliquots were taken out to followthe reaction progress by HPLC. When all GLN was converted to NH₄GLA,product solution was decanted, 8 mL deionized water, 4 mL 1M KHCO₃, and0.4 mL GLN were added to biocatalyst, pH adjusted to pH 7.6, and a newbiocatalyst recycle was repeated with GLN addition up to 3 M GLN at arate of GLN consumption. The initial rate of NH₄GLA synthesis for thefirst biocatalyst recycle (3 M GLN total per recycle) was 144 mM/h.Percent decrease in initial rate of NH₄GLA synthesis vs the recyclenumber are shown in Table 10 (“0.2 M Continuous”).

TABLE 10 Percent decrease in initial rate of NH₄GLA synthesis vs.recycle number at 3 M GLN, 1 M GLN, addition of 3 M GLN in three 1 Mincrements, and at continuous addition of GLN starting with 0.2 M GLN(nd = not determined). Reaction # 3 M 0.2 M (reactions 2-19 are GLN 1 MContinuous recycle reactions (% GLN (1 M + 1 M + Feed with the sameinitial (% initial 1 M) = 3 M GLN catalyst as in reaction reaction GLN(% initial (% initial reaction 1) rate) rate) reaction rate) reactionrate) 1 100 100  100  100 2 62 nd 89 95 3 nd 75 nd 56 4 7 95 88 52 5 ndnd 6 78 nd 7 nd 59 8 79 9 nd 10 nd 11 nd 12 nd 13 44 14 nd 15 nd 16 nd17 nd 18 29 19 25

Example 26 Preparation of GA/PEI-Crosslinked Carrageenan/E. coliFM5/pNM18-210A Beads Comprised of Different Levels of Cross-Linking

The plasmid pNM18-210A, which expresses the nitrilase mutant 210Ala (SEQID NO: 34) from the plasmid pTrcHis2-TOPO® was used to transform E. coliFM5, to generate the strain identified as FM5/pNM18-210A. This strainwas grown in a 10-L fermentation as previously described (see Example 8of U.S. Ser. No. 10/919,182; herein incorporated by reference), and thecell paste was used in a process to convert GLN to glycolic acid (GLA)as follows.

E. coli FM5/pNM18-210A cells were first immobilized in carrageenan beadsaccording to the following procedure. With rapid stirring, 12 g ofcarrageenan (FMC GP911) was slowly added to 228 g deionized distilledwater at 50° C., the resulting mixture heated to 80° C. untilcarrageenan was completely dissolved, and the resulting solution cooledwith stirring to 52° C. In a separate beaker equipped with stir bar,74.9 g of frozen E. coli FM5/pNM18-210A cells (26.7% dcw) was added to85.1 g of 0.35 M Na₂HPO₄ (pH 7.3) at ca. 25° C. and mixed until thecells were suspended, then a deoxyribonuclease I solution (10 μL of12,500 U/mL DNase (Sigma)/100 mL of cell suspension) was added. The cellsuspension was filtered consecutively through a 230 micron and 140micron Nupro TF strainer element filter, and heated with stirring to 50°C. immediately before addition to carrageenan solution. With stirring,160.0 g of E. coli FM5/pNM18-210A cell suspension at 50° C. was added tothe carrageenan solution at 52° C., and the resulting cell/carrageenansuspension was pumped through an electrically-heated 20 gauge needle at47° C. and dripped into 0.25 M KHCO₃ (pH=7.3) with stirring at roomtemperature (ca. 21-22° C.); the flow rate through the needle was set at5-8 mL/min. The resulting beads were allowed to harden for 1 h withstirring, and were stored in 0.25 M KHCO₃ (pH 7.3). Chemicalcrosslinking of the beads was performed by either addition of 0.5 g(hereinafter referred to as “Biocatalyst 1”) or 2.0 g (hereinafterreferred to as “Biocatalyst 2”) of 25% glutaraldehyde (GA) in water(Sigma M 752-07) to 20 g beads suspended in 48 mL of 0.25 M KHCO₃ (pH7.3), and stirring for 1 h at room temperature. To the suspension ofbeads was then added either 2.0 g (Biocatalyst 1) or 4.0 g (Biocatalyst2) of 12.5 wt % polyethylenimine (PEI, BASF LUPASOL PS) in water, andmixing for an additional 18 h at room temperature. TheGA/PEI-crosslinked beads were stored in 1.0 M NH₄HCO₃ (pH 7.3) at 5° C.

Biocatalytic conversion of GLN to GLA was followed by HPLC. Aliquots(0.2 mL) of the reaction mixture were added to 0.01 mL 6 M HCl and 0.8mL of 0.25 M n-propanol in water (HPLC external standard), and analyzedby HPLC (HPX 87H column, 30 cm×7.8 mm; 0.01 NH₂SO₄ mobile phase; 1.0mL/min flow at 50° C.; 10 μL injection volume; RI detector, 20 minanalysis time) for GLN and GLA. The nitrilase activity of theGA/PEI-crosslinked carrageenan/5% (dcw) E. coli FM5/pNM18-210A beads forboth Biocatalyst 1 and Biocatalyst 2, was ˜13 U/g beads, where 1 unit(U) converts 1 μmol of GLN to GLA in 1 min at 25° C.

Example 27 Comparative Conversion of Glycolonitrile (GLN) to AmmoniumGlycolate (NH₄GLA) without Additives in Air

A 50-mL jacketed reaction vessel with overhead stirring was charged with4 g Biocatalyst 1, 12.42 mL deionized water, 0.5 mL 4 M NH₄GLA, and 1.78mL GLN (ca. 52 wt % in water (Fluka)), 1 M GLN final concentration, atpH 7.6, the mixture stirred at 25° C., and 0.2 mL aliquots were takenout to follow the reaction progress by HPLC. When all GLN was convertedto NH₄GLA, second portion of 1.78 mL GLN was added, pH was adjusted topH 7.6 with ammonium hydroxide, and when all GLN was consumed, the thirdportion of 1.78 mL GLN was added, pH adjusted to pH 7.6, and reactioncompleted yielding 3.1 M NH₄GLA solution. Product solution was decanted,12.42 mL deionized water and 1.78 mL GLN were added to biocatalyst, pHadjusted to pH 7.6, GLN conversion proceeded to completion, and additionof GLN, pH adjustment, and completion of GLN conversion were repeatedtwice more to finish the recycle (step-wise conversion of GLN in three1M increments), and biocatalyst recycles were repeated. Percent decreasein initial rate of conversion of the first 1 M GLN solution in therecycle vs recycle number are shown in Table 11 (recycle reactions arereactions 2 through 8).

Example 28 Conversion of Glycolonitrile (GLN) to Ammonium Glycolate(NH₄GLA) without Additives in Oxygen-Free Environment

A 50-mL jacketed reaction vessel with overhead stirring under nitrogenwas charged with 4 g Biocatalyst 1, 12.42 mL deionized water, 0.5 mL 4 MNH₄GLA, and 1.78 mL GLN (ca. 52 wt % in water (Fluka)), 1 M GLN finalconcentration, at pH 7.6, the mixture stirred at 25° C., and 0.2 mLaliquots were taken out to follow the reaction progress by HPLC. Whenall GLN was converted to NH₄GLA, 1.78 ml GLN and 0.2 mL water wereadded, pH was adjusted to pH 7.6 with ammonium hydroxide, and when allGLN was consumed, the third portion of 1.78 mL GLN and 0.2 mL deionizedwater were added, pH adjusted to pH 7.6, and reaction completed,yielding 3.1 M NH₄GLA solution. Product solution was decanted, 12.62 mLdeionized water and 1.78 mL GLN were added to biocatalyst, pH adjustedto pH 7.6, GLN conversion proceeded to completion, and addition of 1.78mL GLN and 0.2 mL deionized water, pH adjustment, conversion of GLN tocompletion were repeated twice more to finish the recycle (step-wiseconversion of GLN in three increments), and biocatalyst recycles wererepeated. The percent decreased in the initial rate for conversion ofthe first 1 M GLN solution in a recycle vs the recycle number are shownin Table 11 (recycle reactions are reactions 2 through 8).

Example 29 Converting Glycolonitrile (GLN) to Ammonium Glycolate(NH₄GLA) in the Presence of Thiosulfate or Dithionite in Oxygen-FreeEnvironment

Biocatalyst recycles were performed as described in Example 28 exceptinstead of 12.42 mL of deionized water, 12.22 mL of deionized water and0.2 mL of 1 M solution of additive (potassium thiosulfate, K₂S₂O₃ orsodium dithionite, K₂S₂O₄) in water were added to start the recycle, andinstead of 0.2 mL water, 0.2 mL 1 M solution of additive in water wasadded to reaction vessel along with each addition of 1.78 mL GLN.Percent decrease in initial rate for conversion of the first 1 M GLNsolution in a recycle vs the recycle number are shown in Table 11(recycle reactions are reactions 2 through 8).

TABLE 11 Percent decrease in initial rate of NH₄GLA synthesis forconversion of the first 1 M GLN solution in a recycle reaction (3 M GLNtotal per recycle) vs the recycle reaction number after addition ofthiosulfate or dithionite to the reaction under nitrogen, or for GLNconversion without additives under nitrogen or in air (nd = notdetermined). K₂S₂O₃/N₂ Na₂S₂O₄/N₂ control/N₂ control/air (% initial (%initial (% initial (% initial reaction reaction reaction reactionReaction # rate) rate) rate) rate) 1 100 100 100 100 2 114 96 77 116 3104 91 97 81 4 101 76 112 nd 5 104 72 74 66 6 102 79 74 nd 7 86 64 63 78 92 74 68

Example 30 Conversion of Glycolonitrile (GLN) to Ammonium Glycolate(NH₄GLA) without Additives in Air at pH 6.0

A 50-mL jacketed reaction vessel with overhead stirring was charged with4 g Biocatalyst 1, 12.42 mL deionized water, 0.5 mL 4 M NH₄GLA, and 1.78mL GLN (ca. 52 wt % in water (Fluka)), 1 M GLN final concentration, atpH 6.0, the mixture stirred at 25° C., and 0.2 mL aliquots were takenout to follow the reaction progress by HPLC. When all GLN was convertedto NH₄GLA, second portion of 1.78 mL GLN was added, pH was adjusted topH 6.0 with ammonium hydroxide, and when all GLN was consumed, the thirdportion of 1.78 mL GLN was added, pH adjusted to pH 6.0, and reactioncompleted yielding 3.1 M NH₄GLA solution. Product solution was decanted,12.42 mL deionized water and 1.78 mL GLN were added to biocatalyst, pHadjusted to pH 6.0, GLN conversion proceeded to completion, and additionof GLN, pH adjustment, and completion of GLN conversion were repeatedtwice more to finish the recycle (step-wise conversion of GLN in threeincrements), and biocatalyst recycles were repeated. Decrease in initialrate of conversion of the first 1 M GLN solution in the recycle vsrecycle number for Biocatalyst 1 are shown in Table 12 (recyclereactions are reactions 2 through 4)

TABLE 12 Percent decrease in initial rate of NH₄GLA synthesis forconversion of first 1 M GLN solution in a recycle (3 M GLN total perrecycle) vs the recycle number at pH 6.0 for Biocatalyst 1. Biocatalyst1 (E. coli FM5/pNM18-210A) pH 6.0 Reaction # (% initial reaction rate) 1100 2 71 3 50 4 24

Example 31 Conversion of Glycolonitrile (GLN) to Ammonium Glycolate(NH₄GLA) Using Immobilized E. coli MG1655/ISW138 Cells Expressing A.facilis 72W Nitrilase at Various Reaction pHs

A 50-mL jacketed reaction vessel equipped with overhead stirring andtemperature control was charged with 4 g of GA/PEI-crosslinkedcarrageenan beads (prepared using the process as described in Example21), washed twice for 15 min with 72 mL of 0.1 M NH₄GLA (pH 7.0))containing 5% (dcw) E. coli MG1655/pSW138 expressing the A. facilis 72Wnitrilase (SEQ ID NO: 6). To the vessel was then added 10.88 g ofdistilled water and 2.98 mL 4.0 M NH₄GLA (pH 7.5), the appropriateamounts of either 70 wt % glycolic acid (GLA) (Aldrich) or 1:4 dilutionof ammonium hydroxide (28-30 wt %) in water (Table 13), and the reactionvessel was flushed with nitrogen. The mixture was stirred at 25° C. and2.15 mL of 49.88 wt % glycolonitrile (GLN) in water (2.25 g, 19.6 mmol(Fluka)) was added to yield 1 M GLN at pH 4.0, 4.7, 5.5, 6.7, or 7.5(Table 14).

Four 0.100-mL reaction samples were removed at predetermined times afterthe GLN addition and analyzed by HPLC to determine the initial reactionrate. The initial reaction rates as an average of rates in duplicateruns at each pH are listed in Table 14.

TABLE 13 Amounts of 70 wt % glycolic acid in water (GLA) or 1:4 dilutionof 28-30 wt % ammonium hydroxide (NH₄OH) in water used to preparereaction solutions of indicated pH. Aqueous Aqueous pH GLA (mL) NH₄OH(mL) 4.0 0.700 0 4.7 0.150 0 5.5 0 0 6.7 0 0.050 7.5 0 0.100

TABLE 14 Initial reaction rate for conversion of GLN to NH₄GLA usingimmobilized E. coli MG1655/pSW138 cells expressing A. facilis 72Wnitrilase at various pHs (average of duplicate reactions). InitialReaction Rate pH (mM GLA/h) 4.0 0 4.7 68 5.5 347 6.7 354 7.5 351

Example 32 Hydrolysis of Glycolonitrile to Ammonium Glycolate UsingImmobilized E. coli FM5/pNM18 Expressing A. facilis 72W Nitrilase in thePresence or in the Absence of Hydrogen Cyanide (HCN)

A 50-mL jacketed reaction vessel equipped with overhead stirring andtemperature control was charged with 4 g of GA/PEI-crosslinkedcarrageenan beads (prepared using the process described in Example 21),washed twice for 15 min with 72 mL of 0.1 M NH₄GLA (pH 7.5)) containing5% (dcw) E. coli FM5/pNM18 expressing the A. facilis 72W nitrilase (SEQID NO: 6). To the vessel was then added 10.9 g of distilled water and3.0 mL 4.0 M NH₄GLA (pH 7.5), and the reaction vessel was flushed withnitrogen. The mixture was stirred at 25° C. and an aliquot of 1.777 mLof 60.51 wt % glycolonitrile (GLN) in water (1.885 g, 20.0 mmol (Fluka,redistilled)) with or without 0.063 mL 50 wt % HCN in water (0.054 g, 1mmol) was first added, followed immediately by addition of 0.320 mL of a1:16 dilution of ammonium hydroxide (28-30 wt %) in water. Four 0.100-mLreaction samples were removed at predetermined times after the first GLNaddition and analyzed by HPLC to determine the initial reaction rate. Atcompletion of GLN conversion, the second aliquot each of GLN andammonium hydroxide was added to maintain the concentration of GLN at ≦1M and pH within a range of 7.0-7.5, and after the GLN conversion wascompleted, the third aliquot of each of GLN and ammonium hydroxide wasadded. At completion of the reaction, there was 100% conversion of GLNto produce glycolic acid (as the ammonium salt) in >99% yield, and theconcentration of ammonium glycolate produced from added GLN wasapproximately 2.5 M (3.0 M total ammonium glycolate, including initialammonium glycolate buffer, in a final reaction volume of ca. 23.7 mL).

At the end of the first reaction, the aqueous product mixture wasdecanted from the catalyst (under nitrogen), to the reaction vessel thenadded 13.9 mL of distilled, deionized water, and a second reaction wasperformed at 25° C. by the addition of aliquots of aqueous GLN andammonium hydroxide as described immediately above. The initial reactionrates for consecutive batch reactions with catalyst recycle are listedin Table 15 (recycle reactions are reactions 2 through 9).

TABLE 15 Initial reaction rate for conversion of GLN to GLA inconsecutive batch reactions with catalyst recycle using immobilized E.coli FM5/pNM18 expressing A. facilis 72W nitrilase in the presence andin the absence of HCN. No HCN additive Rxn # (mM GLA/h) (mM GLA/h) 1 260183 2 289 269 3 291 218 4 271 222 5 238 235 6 257 240 7 250 208 8 239177 9 213 188

Example 33 Affect of Addition of Either Formaldehyde or Hydrogen Cyanidein Consecutive Batch Reactions for Hydrolysis of Glycolonitrile toAmmonium Glycolate Using Immobilized E. coli FM5/pNM18 Expressing the A.facilis 72W Nitrilase

The reactions were run, characterized, and the biocatalyst was recycledas described in Example 32 for reactions without addition of HCN, exceptthat each aliquot of 1.777 mL of 60.51 wt % glycolonitrile (GLN) inwater (1.885 g, 20.0 mmol (Fluka, redistilled)) contained either 0.074mL 37 wt % HCHO in water (0.081 g, 1 mmol) (recycles 1, 2, 3, and 6) or0.063 mL 50 wt % HCN in water (0.054 g, 1 mmol) (recycles 4, 5, and 7)(Table 16). The data for reactions without addition of HCHO or HCN arerepeated from Table 15 for comparison.

TABLE 16 Initial reaction rate for conversion of GLN to GLA inconsecutive batch reactions with catalyst recycle using immobilized E.coli FM5/pNM18 expressing A. facilis 72W nitrilase at addition of HCHO(reactions 1, 2, 3, and 6) or addition of HCN (reactions 4, 5, and 7) tothe same reaction series, and without addition of HCHO or HCN. HCHO HCNNo additives Initial rate Initial rate initial rate Reaction # (mMGLA/h) (mM GLA/h) (mM GLA/h) 1 192 183 2 152 269 3 90 218 4 234 222 5218 235 6 113 240 7 219 208 8 177 9 188

Example 34 Hydrolysis of Glycolonitrile to Ammonium Glycolate Using E.coli FM5/pNM18-L201Q cells Expressing A. facilis 72W Nitrilase MutantL201Q

A 50-mL centrifuge tube was charged with 1.25 mL uniform suspensionobtained using 6 g of E. coli FM5/pNM18-L201Q expressing the A. facilis72W nitrilase mutant L201Q (SEQ ID NO: 8) and 7.54 mL 0.35 M Na₂HPO₄ (pH7.5), 35 mL 0.35 M Na₂HPO₄ (pH 7.5) was added, the tube was centrifugedat 5000 rpm for 20 min, the supernatant was carefully and fully removedfrom cell paste, and 935 mg of the centrifuged cell paste wastransferred to a 150-mL jacketed reaction vessel equipped with overheadstirring and temperature control. To the vessel was then added 52.54 mLof 0.3 M NH₄GLA (pH 7.5), 7.88 mL 4.0 M NH₄GLA (pH 7.5), and 9.63 mL ofdistilled water, and the reaction vessel was flushed with nitrogen. Themixture was stirred at 25° C., 7.82 mL of 54.61 wt % glycolonitrile(GLN) in water (8.18 g, 78.3 mmol (Fluka)) was added, and pH wasadjusted to pH 7.5 by a 1:4 dilution of ammonium hydroxide (28-30 wt %)in water. To determine the initial reaction rates, four 0.050-mLreaction samples were removed at predetermined times after the first GLNaddition, added to assay mix (0.025 mL of 6.0 N HCl and 0.800 mL 0.18 Mn-propanol), vortexed, centrifuged at 12,000 rpm for 6 min, andsupernate was analyzed by HPLC as described in Example 12. At completionof GLN conversion, the second aliquot of GLN was added, pH was adjustedto 7.5 with ammonium hydroxide, and after the GLN conversion wascompleted, the third GLN aliquot was added and pH was adjusted to pH7.5. At completion of the reaction, there was 100% conversion of GLN toproduce glycolic acid (as the ammonium salt) in >99% yield, and theconcentration of ammonium glycolate produced from added GLN wasapproximately 2.5 M (2.9 M total ammonium glycolate, including initialammonium glycolate buffer, in a final reaction volume of ca. 94.05 mL).

At the end of the first reaction, the aqueous product mixture wascentrifuged from the cell pastes (5000 rpm, 20 min). The cell paste wasweighed and transferred back to the reaction vessel. To the vessel thenadded 52.54 mL 0.3 M NH₄GLA (pH 7.5), 7.88 mL 4.0 M NH₄GLA (pH 7.5) and9.63 mL of distilled water, the reaction vessel was flushed withnitrogen, and a second reaction was performed at 25° C. by the additionof aliquots of aqueous GLN and ammonium hydroxide as describedimmediately above. The weight of cell paste recovered after reaction 4by centrifugation of reaction solution as described above was 964 mg.

The initial reaction rates for consecutive batch reactions with catalystrecycle are listed in Table 17 (recycle reactions are reactions 2through 4).

TABLE 17 Initial reaction rate for conversion of GLN to GLA inconsecutive batch reactions with catalyst recycle using E. coliFM5/pNM18-L201Q cell paste expressing A. facilis 72W nitrilase mutantL201Q. Initial Rate Reaction # (mM GLA/h) 1 180 2 157 3 147 4 133

Example 35 Hydrolysis of Glycolonitrile to Ammonium Glycolate UsingImmobilized E. coli MG1655/ISW138 Transformants Expressing A. facilis72W Nitrilase or A. facilis 72W Nitrilase Mutants

A 50-mL jacketed reaction vessel equipped with overhead stirring andtemperature control was charged with 8 g of GA/PEI-crosslinkedcarrageenan beads (prepared using the process as described in Example21), washed twice for 15 min with 72 mL of 0.1M NH₄GLA (pH 7.0))containing 5% (dcw) E. coli MG1655/pSW138 transformant expressing the A.facilis 72W nitrilase (SEQ ID NO: 6), or the A. facilis 72W nitrilasemutants F168V (SEQ ID NO: 32), F168M (SEQ ID NO: 28), F168K (SEQ ID NO:26), F168T (SEQ ID NO: 30), and L201Q (SEQ ID NO: 8). To the vessel wasthen added 14.632 g of distilled water and 6.0 mL 4.0 M NH₄GLA (pH 7.0),and the reaction vessel flushed with nitrogen. The mixture was stirredat 25° C. while programmable syringe pumps were used to add eightaliquots of 1.08 mL of 59 wt % glycolonitrile (GLN) in water (1.14 g,12.0 mmol (Fluka, redistilled)) and 0.288 mL of a 1:16 dilution ofammonium hydroxide (28-30 wt %) in water (2.304 mL total); one aliquoteach of GLN and ammonium hydroxide was added simultaneously every 2 h tomaintain the concentration of GLN at ≦400 mM and the pH within a rangeof 6.5-7.5. Four 0.050-mL reaction samples were removed at predeterminedtimes after the first GLN addition and analyzed by HPLC to determine theinitial reaction rate. At completion of the reaction, there was 100%conversion of GLN to produce glycolic acid (as the ammonium salt)in >99% yield, and the concentration of ammonium glycolate produced fromadded GLN was approximately 2.4 M (3.0 M total ammonium glycolate,including initial ammonium glycolate buffer, in a final reaction volumeof ca. 39.5 mL).

At the end of the first reaction, the aqueous product mixture wasdecanted from the catalyst (under nitrogen), leaving ca. 10.3 g of amixture of immobilized cell catalyst (8.0 g) and remaining productmixture (ca. 2.3 g). To the reaction vessel then added 18.3 mL ofdistilled, deionized water, and a second reaction was performed at 25°C. by the addition of aliquots of aqueous GLN and ammonium hydroxide asdescribed immediately above. The initial reaction rates for consecutivebatch reactions with catalyst recycle are listed in Table 18 (recyclereactions are reactions 2 through 55).

The catalyst productivity (total grams GLA produced/gram dry cell weight(dcw) enzyme catalyst) was calculated for each nitrilase from the totalnumber of consecutive batch reactions with catalyst recycle thatresulted in 100% conversion of glycolonitrile. The catalyst productivityfor each enzyme catalyst was: E. coli MG1655/pSW138-F168V, 1001 g GLA/gdcw (55 consecutive batch reactions); E. coli MG1655/pSW138-F168M, 473 gGLA/g dcw (26 consecutive batch reactions); E. coli MG1655/pSW138-F168K,473 g GLA/g dcw (26 consecutive batch reactions); E. coliMG1655/pSW138-F168T, 364 g GLA/g dcw (20 consecutive batch reactions);E. coli MG1655/pSW138-L201Q, 346 g GLA/g dcw (19 consecutive batchreactions); E. coli MG1655/pSW138, 182 g GLA/g dcw (10 consecutive batchreactions).

TABLE 18 Initial reaction rates for conversion of GLN to GLA inconsecutive batch reactions with catalyst recycle using immobilized E.coli MG1655/pSW138 transformants expressing A. facilis 72W nitrilase orA. facilis 72W nitrilase mutants (nd = not determined). MG1655/ MG1655/MG1655/ MG1655/ MG1655/ pSW138- pSW138- pSW138- pSW138- pSW138- MG1655/F168V F168M F168K F168T L201Q pSW138 Rxn # (mM GLA/h) (mM GLA/h) (mMGLA/h) (mM GLA/h) (mM GLA/h) (mM GLA/h) 1 1050 658 652 423 757 330 2 698538 560 389 611 220 3 719 488 394 453 497 207 4 654 452 nd 311 435 174 5719 441 322 494 549 193 6 559 378 439 456 416 171 7 531 258 435 408 482207 8 634 407 209 378 453 141 9 586 340 294 502 653 123 10 609 303 420468 537 92 11 432 313 426 428 443 76 12 397 560 361 481 388 13 318 430422 391 359 14 449 391 387 255 302 15 259 304 444 260 246 16 370 308 452362 377 17 401 330 448 318 307 18 579 384 nd 299 252 19 392 253 nd 233282 20 nd nd 487 372 209 21 nd nd nd nd nd 22 356 247 nd 144 116 23 ndnd 355 165 129 24 nd nd 300 25 402 219 252 26 407 134 270 27 390 87 14128 280 45 29 297 24 30 277 31 300 32 325 33 344 34 340 35 317 36 277 37191 38 279 39 325 40 372 41 257 42 329 43 530 44 235 45 291 46 339 47226 48 309 49 406 50 456 51 242 52 168 53 217 54 220 55 59

Example 36 Characterization of Ammonium Glycolate Obtained by Conversionof GLN Using Immobilized E. coli MG1655/ISW138 Transformant ExpressingA. facilis 72W Nitrilase Mutant F168V

To evaluate composition of product solutions obtained by GLN (Fluka,redistilled) hydrolysis with immobilized MG1655/pSW138-F168V biocatalyst(see Example 35, Table 18), the product solutions produced in reactions5, 10, and 38 were characterized by HPLC and ¹H NMR spectroscopy.Concentration of glycolate determined by HPLC was 3.1 M. Quantitative ¹HNMR spectra were obtained using a Varian Unity Inova spectrometer(Varian, Inc., Palo Alto, Calif.) operating at 500 MHz. Samples wereprepared by adding 150 μL of the reaction product along with 400 μL ofD₂O to a 5 mm NMR tube.

¹H NMR spectra were acquired using a spectral width of 6000 Hz with thetransmitter located at 5 ppm, and a 90-degree pulse (5.9 microseconds ata transmitter power of 49 db). An acquisition time of 4 seconds was usedwhich led to a total data size of 48,000 points. The longest ¹HT₁ (8sec) was associated with the methanol CH₃ protons, and the total delaytime prior to acquisition was therefore set to 50 seconds (i.e., morethan 5 times the methanol CH₃T₁). This pre-delay time was split betweena simple delay time (“d1”) and a solvent saturation pulse of 30 secondsapplied on resonance for residual water at a transmitter power of −6 db.Signal averaging of 32 scans was preceded by 4 steady-state (“dummy”)scans to give a total experiment time of approximately 32 minutes.Assignments were obtained by comparison of ¹H NMR chemical shifts withthose obtained in previous 2-dimensional NMR correlation experiments,and by spiking experiments.

The impurities observed in the ammonium glycolate product solutions by¹H NMR spectroscopy were categorized, on the basis of their functionalgroups, into the following functional group categories:formaldehyde-derived, formic acid-derived, methanol-derived, andmethyl-derived. Integrated peak areas of proton signals for each of thecategories were assigned as follows: two protons were assigned toformaldehyde functionality, one proton assigned to formic acidfunctionality, three protons assigned to methanol functionality, andthree protons assigned to methyl functionality. Integrated peak areasfor ammonium glycolate were divided by 2 (the number of ammoniumglycolate protons) and assigned a value of 100%. Integrated peak areasof the protons observed for each of the impurity functional groupcategories were divided by the number of corresponding protons (seeabove), and the resulting integrated peak area divided by the integratedpeak area of one glycolate proton present in the sample to determine thepercent concentration of the impurity relative to the concentration ofammonium glycolate present. The yield of ammonium glycolate (based on100% conversion of GLN) and the % purity of ammonium glycolate (based onrelative concentration of glycolate and total impurities) is listed inTable 19.

TABLE 19 Yield and purity of ammonium glycolate produced by theconversion of GLN using immobilized E. coli MG1655/pSW138 transformantexpressing A. facilis 72W nitrilase mutant F168V. ammonium glycolateammonium glycolate Reaction # yield (%) purity (%) Example 26, reaction5 99% 98.5 Example 26, reaction 10 99% 98.5 Example 26, reaction 38 99%98.8

Example 37 Hydrolysis of Glycolonitrile Obtained from Hydrogen Cyanideand Formaldehyde to Ammonium Glycolate using Immobilized E. coliMG1655/ISW138 Transformant Expressing A. facilis 72W Nitrilase MutantsF168V

Glycolonitrile used for the reactions below was prepared as described inExamples 4-8, except the experimental set-up was modified to avoid theraising of the reaction flask and corresponding bath/stir plate assemblyat the start of the HCHO feed into the reactor. The outlet of the heatedsection of the HCHO feed line was directly connected to an insulated3-way ball valve which, in turn, could direct the HCHO feed throughseparate lines into either the reaction vessel or an externalscintillation vial. At the beginning of the synthesis procedure, theHCHO feed was directed by the 3-way valve into the scintillation vial.Upon the apparent onset of 2-phase flow of the HCHO feed, the 3-wayvalve was turned to direct the HCHO feed into the reaction flask.

Biotransformation reactions were run as described in Example 35 exceptthat the reaction volume was one half of the reactions in Example 35 andpH was maintained at pH 7.5. To a reaction vessel were added 4 gbiocatalyst beads, 7.32 g of distilled water, and 3 mL 4.0 M NH₄GLA (pH7.5), the vessel was flushed with nitrogen, the mixture was stirred at25° C., and programmable syringe pumps were used to add eight aliquotsof 0.54 mL of 60 wt. % glycolonitrile (GLN) in water (0.58 g, 6.0 mmol(GLN prepared as described above) and 0.15 mL of a 1:16 dilution ofammonium hydroxide (28-30 wt %) in water (1.2 mL total); one aliquoteach of GLN and ammonium hydroxide was added simultaneously every 2 h.At the end of the first reaction, the aqueous product mixture wasdecanted from the catalyst (under nitrogen), 10.32 mL of deionized waterwas added, and a new biocatalyst recycle was started with addition ofGLN and ammonium hydroxide as described immediately above. The initialreaction rates for consecutive batch reactions with catalyst recycle arelisted in Table 20 (recycle reactions are reactions 2 through 24). Forall reactions, the conversion of GLN to GLA was 100%, and the yield ofGLA was greater than 99%.

TABLE 20 Initial reaction rates for conversion of GLN produced (asdescribed in Example 37) to GLA in consecutive batch reactions withcatalyst recycle using immobilized E. coli MG1655/pSW138 transformantexpressing A. facilis 72W nitrilase mutant F168V (nd = not determined).Initial rate Rxn # (mM GLA/h) 1 1177  2 771 3 514 4 526 5 nd 6 425 7 4408 386 9 434 10 329 11 469 12 396 13 358 14 318 15 387 16 365 17 nd 18387 19 415 20 nd 21 nd 22 370 23 nd 24 260

Example 38 Isolation of Glycolic Acid from Ammonium Glycolate byFixed-Bed Ion Exchange Chromatography

GLN synthesized from hydrogen cyanide and formaldehyde as described inExamples 4-8 (synthesis of high purity GLN), was converted to ammoniumglycolate as described in Example 22, without additives (except that thereaction volume was scaled-up 18-fold), and fixed-bed ion exchange wasused to convert the ammonium glycolate product solution to glycolicacid.

A 5 cm ID×60 cm borosilicate glass column (Spectrum-Chromatography)fitted with Teflon® PTFE end caps and Dowex® G-26 strongly acidic cationresin in the H⁺ form (Dow Chemical Co) were used. A 5-gallonpolypropylene feed jug was used to supply ultrapure water (18+MΩ,produced by a Sybron-Barnstead Nanopure II unit) to the column feed pump(Cole-Parmer variable-speed diaphragm pump with all-Teflon® head) forresin pre- and post-rinsing. The jug was nitrogen-purged at all times toprevent absorption of atmospheric CO₂. After pre-rinsing the filledcolumn (initial height=23″, bed volume=1147 mL) with ultrapure water toan effluent of >5 MΩ, the ammonium glycolate (1.3 liters (1428 g),pH=7.09) was pumped through the bed upflow at 40 mL; when the glycolatewas depleted, the unit was switched back to ultrapure water pumping atthe same rate to continue pushing the feed material through the bed.During the run the column effluent was captured continuously in 50 mLincrements using pre-rinsed HDPP (high density polypropylene) samplebottles; a total of thirty-eight 50-mL samples of effluent were takencontinuously and were analyzed for pH (pH meter), glycolateconcentration (HPLC), and ammonium ion content (via ion chromatography).

Determination of ammonium ion content was done using Dionex IP25 pumpequipped with CD20 conductivity detector and Dionex CS17 column (3-11 mMMethane Sulfonic Acid, 1.0 mL/min, suppressed with a Dionex CSRS ultraset to 100 microamp, 1.0 mL/min, 100 microliters sample loop), andcation 3-11 mM CS17 method was applied for the analysis.

Fractions 12 to 23 were combined (600 mL total), stirred overnight with5 g fresh Dowex® G-26 resin (pre-rinsed 3 times with 45 mL of deionizedwater for 20 min), 651 g of glycolic acid solution was collected byfiltration. The solution was concentrated by rotary evaporation toproduce 70 wt % glycolic acid (140 g product). Analysis of the 70 wt %glycolic acid for impurities indicated the purity of glycolic acid wasgreater than 99.9%.

Example 39 Solvent Extraction Using Approximately 70% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and 20%Kerosene at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 70% (volume/volume) of trialkyl amine(Alamine® 336; Cognis Corp., Cincinnati, Ohio), 10% (volume/volume)methyl isobutyl ketone (MIBK) and 20% (volume/volume) kerosene. The pHof an aqueous solution of ammonium glycolate (5 wt % to 40 wt %) wasadjusted to about pH 2 to 3 with concentrated sulfuric acid (H₂SO₄),then 1 mL of the resulting aqueous solution was added to the reactor.The resulting mixture was stirred for 30 minutes at 25° C. The stirringwas stopped and the two phases allowed to separate, then the organic andaqueous phases were each sampled and analyzed for glycolic acidconcentration by HPLC. For each initial glycolic acid concentration,Table 21 lists the final concentration of glycolic acid in each phase ofthe resulting mixture, and the partition coefficient calculated for eachinitial glycolic acid concentration.

TABLE 21 Partition coefficient Initial wt % Final wt % Final wt % ofglycolic acid glycolic acid in glycolic acid in glycolic acid in(organic wt %/ aqueous phase aqueous phase organic phase aqueous wt %)3.6 1.9 2.0 1.1 8.5 4.5 4.8 1.1 12.1 6.5 6.6 1.0 16.1 9.2 8.6 0.94 20.610.9 9.8 0.90 25.1 15.4 13.1 0.85 29.1 19.6 14.2 0.73 32.8 22.9 15.40.67

Example 40 Solvent Extraction Using Approximately 70% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and 20%Kerosene at 50° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 70% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) methyl isobutyl ketone (MIBK) and 20%(volume/volume) kerosene. The pH of an aqueous solution of ammoniumglycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 50° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 22 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 22 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 3.4 1.1 0.32 8.5 6.3 4.10.66 12.1 8.2 5.2 0.63 16.1 10.5 7.7 0.74 20.6 13.2 9.0 0.69 25.1 16.513.8 0.83 29.1 19.8 13.4 0.68 32.8 23.5 14.4 0.61

Example 41 Solvent Extraction Using Approximately 70% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and 20%Kerosene at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 70% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) methyl isobutyl ketone (MIBK) and 20%(volume/volume) kerosene. The pH of an aqueous solution of ammoniumglycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 75° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 23 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 23 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 3.9 0.6 0.16 8.5 7.6 1.70.22 12.1 9.8 3.4 0.35 16.1 12.6 5.0 0.40 20.6 15.5 7.5 0.49 25.1 18.310.2 0.56 29.1 22.7 12.3 0.54 32.8 26.3 13.7 0.52

Example 42 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis) and 10% (volume/volume) methyl isobutyl ketone (MIBK). The pHof an aqueous solution of ammonium glycolate (5 wt % to 40 wt %) wasadjusted to pH 2 to 3 with concentrated sulfuric acid, then 1 mL of theresulting aqueous solution was added to the reactor. The resultingmixture was stirred for 30 minutes at 25° C. The stirring was stoppedand the two phases allowed to separate, then the organic and aqueousphases were each sampled and analyzed for glycolic acid concentration byHPLC. For each initial glycolic acid concentration, Table 24 lists thefinal concentration of glycolic acid in each phase of the resultingmixture, and the partition coefficient for each initial glycolic acidconcentration.

TABLE 24 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 1.8 3.6 1.98 8.5 4.3 6.81.57 12.1 5.9 8.1 1.38 16.1 8.4 12.2 1.44 20.6 12.6 14.2 1.13 25.1 13.616.2 1.19 29.1 16.6 18.9 1.14 32.8 21.1 19.4 0.92

Example 43 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis) and 10% (volume/volume) methyl isobutyl ketone (MIBK). The pHof an aqueous solution of ammonium glycolate (5 wt % to 40 wt %) wasadjusted to pH 2 to 3 with concentrated sulfuric acid, then 1 mL of theresulting aqueous solution was added to the reactor. The resultingmixture was stirred for 30 minutes at 75° C. The stirring was stoppedand the two phases allowed to separate, then the organic and aqueousphases were each sampled and analyzed for glycolic acid concentration byHPLC. For each initial glycolic acid concentration, Table 25 lists thefinal concentration of glycolic acid in each phase of the resultingmixture, and the partition coefficient for each initial glycolic acidconcentration.

TABLE 25 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.4 1.8 0.75 8.5 5.6 4.00.70 12.1 7.7 5.8 0.76 16.1 10.9 7.9 0.73 20.6 12.8 10.0 0.79 25.1 16.013.8 0.87 29.1 18.4 15.5 0.84 32.8 21.7 18.6 0.86

Example 44 Solvent Extraction Using Approximately 50% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and 40%Kerosene at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 50% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) methyl isobutyl ketone (MIBK) and 40%(volume/volume) kerosene. The pH of an aqueous solution of ammoniumglycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 25° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 26 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 26 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.2 1.6 0.76 8.5 5.2 3.70.71 12.1 7.7 5.6 0.72 16.1 11.0 7.1 0.65 20.6 13.8 8.1 0.59 25.1 18.59.4 0.51 29.1 21.9 10.5 0.48 32.8 26.1 12.1 0.46

Example 45 Solvent Extraction Using Approximately 50% C8-C10Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and 40%Kerosene at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 50% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) methyl isobutyl ketone (MIBK) and 40%(volume/volume) kerosene. The pH of an aqueous solution of ammoniumglycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 75° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 27 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 27 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.7 1.0 0.38 8.5 6.6 2.10.32 12.1 9.4 3.3 0.35 16.1 13.5 3.8 0.28 20.6 16.2 5.4 0.33 25.1 20.17.0 0.35 29.1 23.9 8.3 0.35 32.8 27.4 9.5 0.35

Example 46 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% 1-Octanol at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) 1-octanol. The pH of an aqueous solutionof ammonium glycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 25° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 28 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 28 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 1.9 2.0 1.09 8.5 4.4 4.61.03 12.1 5.8 7.5 1.30 16.1 8.5 10.2 1.20 20.6 10.4 12.4 1.20 25.1 13.815.2 1.10 29.1 17.2 16.9 0.99 32.8 21.7 17.7 0.82

Example 47 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% 1-Octanol at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) 1-octanol. The pH of an aqueous solutionof ammonium glycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 75° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 29 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 29 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.5 1.4 0.54 8.5 5.9 3.30.56 12.1 8.4 5.2 0.62 16.1 11.3 7.2 0.63 20.6 13.3 9.7 0.73 25.1 16.812.6 0.75 29.1 19.5 14.2 0.73 32.8 22.8 16.1 0.70

Example 48 Solvent Extraction Using Approximately 70% C8-C10Trialkylamine in Combination with 30% 1-Octanol at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 70% (volume/volume) Alamine® 336(Cognis), 30% (volume/volume) 1-octanol. The pH of an aqueous solutionof ammonium glycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 25° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 30 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 30 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 1.8 2.0 1.10 8.5 4.5 4.51.01 12.1 6.8 7.0 1.02 16.1 9.7 8.7 0.90 20.6 12.8 9.8 0.76 25.1 16.911.2 0.67 29.1 20.7 12.3 0.60 32.8 24.9 13.4 0.54

Example 49 Solvent Extraction Using Approximately 70% C8-C10Trialkylamine in Combination with 30% 1-Octanol at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 70% (volume/volume) Alamine® 336(Cognis), 30% (volume/volume) 1-octanol. The pH of an aqueous solutionof ammonium glycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 75° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 31 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 31 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.3 1.6 0.69 8.5 5.5 4.10.74 12.1 8.5 5.4 0.64 16.1 11.0 7.5 0.68 20.6 14.0 8.6 0.62 25.1 18.211.1 0.61 29.1 24.1 12.0 0.50 32.8 25.5 13.4 0.52

Example 50 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% Toluene at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) toluene. The pH of an aqueous solution ofammonium glycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 25° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 32 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 32 Initial wt % Final wt % Final wt % Partition coefficientglycolic acid in glycolic acid in glycolic acid in (wt % org./wt %aqueous phase aqueous phase organic phase aq.) 3.6 1.9 2.4 1.22 8.5 4.66.5 1.41 12.1 6.0 8.9 1.46 16.1 8.8 10.8 1.23 20.6 11.0 13.2 1.20 25.114.4 18.2 1.26 29.1 17.7 17.8 1.00 32.8 23.0 19.8 0.86

Example 51 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% Toluene at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) toluene. The pH of an aqueous solution ofammonium glycolate (5 wt % to 40 wt %) was adjusted to pH 2 to 3 withconcentrated sulfuric acid, then 1 mL of the resulting aqueous solutionwas added to the reactor. The resulting mixture was stirred for 30minutes at 75° C. The stirring was stopped and the two phases allowed toseparate, then the organic and aqueous phases were each sampled andanalyzed for glycolic acid concentration by HPLC. For each initialglycolic acid concentration, Table 33 lists the final concentration ofglycolic acid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 33 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.6 1.3 0.51 8.5 6.0 3.50.58 12.1 8.3 5.5 0.67 16.1 11.9 7.5 0.63 20.6 13.8 9.0 0.65 25.1 16.412.0 0.73 29.1 19.3 14.5 0.75 32.8 22.0 16.6 0.75

Example 52 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% Xylenes at 25° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) xylenes (mixed xylene isomers). The pH ofan aqueous solution of ammonium glycolate (5 wt % to 40 wt %) wasadjusted to pH 2 to 3 with concentrated sulfuric acid, then 1 mL of theresulting aqueous solution was added to the reactor. The resultingmixture was stirred for 30 minutes at 25° C. The stirring was stoppedand the two phases allowed to separate, then the organic and aqueousphases were each sampled and analyzed for glycolic acid concentration byHPLC. For each initial glycolic acid concentration, Table 34 lists thefinal concentration of glycolic acid in each phase of the resultingmixture, and the partition coefficient for each initial glycolic acidconcentration.

TABLE 34 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 1.9 2.5 1.31 8.5 4.4 6.11.39 12.1 5.7 8.0 1.40 16.1 8.2 10.3 1.25 20.6 10.1 12.8 1.27 25.1 15.115.1 1.00 29.1 16.2 22.7 1.40 32.8 20.5 18.6 0.91

Example 53 Solvent Extraction Using Approximately 90% C8-C10Trialkylamine in Combination with 10% Xylenes at 75° C.

Into a 4-mL glass reactor equipped with magnetic stir bar was placed 1mL of a mixed solvent containing 90% (volume/volume) Alamine® 336(Cognis), 10% (volume/volume) xylenes (mixed xylene isomers). The pH ofan aqueous solution of ammonium glycolate (5 wt % to 40 wt %) wasadjusted to pH 2 to 3 with concentrated sulfuric acid, then 1 mL of theresulting aqueous solution was added to the reactor. The resultingmixture was stirred for 30 minutes at 75° C. The stirring was stoppedand the two phases allowed to separate, then the organic and aqueousphases were each sampled and analyzed for glycolic acid concentration byHPLC. For each initial glycolic acid concentration, Table 35 lists thefinal concentration of glycolic acid in each phase of the resultingmixture, and the partition coefficient for each initial glycolic acidconcentration.

TABLE 35 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient aqueous phase aqueousphase organic phase (wt % org./wt % aq.) 3.6 2.6 1.4 0.55 8.5 6.0 3.30.55 12.1 8.4 5.6 0.66 16.1 11.6 7.4 0.64 20.6 14.0 9.1 0.65 25.1 16.412.0 0.73 29.1 19.2 14.4 0.75 32.8 22.5 16.1 0.72

Example 54 Back Extraction Using Water from a Loaded Solvent ofApproximately 70% C8-C10 Trialkylamine in Combination with 10% MethylIsobutyl Ketone and 20% Kerosene

Following the procedures in Example 1, into a 1-L cylindrical glassvessel on an extraction mixer (mix by rotating back and forthvertically) was placed 100 mL of a mixed solvent containing 70%(volume/volume) Alamine® 336 (Cognis), 10% (volume/volume) methylisobutyl ketone (MIBK) and 20% (volume/volume) kerosene. The pH of anaqueous solution of ammonium glycolate (10 wt % to 50 wt %) was adjustedto approximately pH 2 to 3 with concentrated sulfuric acid, then 100 mLof the resulting aqueous solution was added to the extraction mixer. Theresulting mixture was stirred for 60 minutes at room temperature. Themixing was stopped and the two phases allowed to separate, then theorganic and aqueous phases were each sampled and analyzed for glycolicacid concentration by HPLC. The organic phase was collected and used inthe back extraction. This organic phase containing glycolic acid isreferred as “loaded solvent” below.

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL deionized water and 10 mL of the loaded solvent.The vessel was then closed, and the headspace purged with nitrogen. Theresulting mixture was stirred for 60 minutes at 120° C. under 40 psig(˜275.8 kPa) nitrogen. The stirring was stopped and the two phasesallowed to separate at 120° C., then the organic phase was sampled underpressure through the top dip tube into a Hoke cylinder, and aqueousphases was sampled under pressure through the bottom dip tube intoanother Hoke cylinder. Both phases were analyzed for glycolic acidconcentration by HPLC.

For each initial glycolic acid concentration in the loaded solvent,Table 36 lists the final concentration of glycolic acid in each phase ofthe resulting mixture, and the partition coefficient for each initialglycolic acid concentration.

TABLE 36 Initial wt % Final wt % Final wt % Partition coefficientglycolic acid in glycolic acid in glycolic acid in (wt % org./wt %loaded solvent aqueous phase organic phase aq.) 3.9 1.7 0.82 0.47 11.36.5 3.4 0.53 16.0 9.0 4.6 0.51 17.5 9.8 5.1 0.52

Example 55 Back Extraction from a Loaded Solvent of Approximately 70%C8-C10 Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and20% Kerosene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL aqueous solution of glycolic acid (20 wt % or 40wt %) and 10 mL of the loaded solvent (see Example 54). The vessel wasthen closed, and the headspace purged with nitrogen. The resultingmixture was stirred for 60 minutes at 120° C. under 40 psig (˜275.8 kPa)nitrogen. The stirring was stopped and the two phases allowed toseparate at 120° C., then the organic phase was sampled under pressurethrough the top dip tube into a Hoke cylinder, and aqueous phases wassampled under pressure through the bottom dip tube into another Hokecylinder. Both phases were analyzed for glycolic acid concentration byHPLC.

For each initial glycolic acid concentration in the loaded solvent andaqueous solution, Table 37 lists the final concentration of glycolicacid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 37 Initial wt % Initial wt % Final wt % Partition glycolic acidglycolic acid glycolic acid Final wt % coefficient in aqueous in loadedin aqueous glycolic acid in (wt % org./ phase solvent phase organicphase wt % aq.) 20.0 16.0 20.7 12.1 0.59 40.0 17.5 33.7 17.3 0.51

Example 56 Back Extraction Using Water from a Loaded Solvent ofApproximately 70% C8-C10 Trialkylamine in Combination with 10% MethylIsobutyl Ketone and 20% Kerosene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL deionized water and 10 mL of the loaded solvent(see Example 54). The vessel was then closed, and the headspace purgedwith nitrogen. The resulting mixture was stirred for 60 minutes at 140°C. under 40 psig (˜275.8 kPa) nitrogen. The stirring was stopped and thetwo phases allowed to separate at 140° C., then the organic phase wassampled under pressure through the top dip tube into a Hoke cylinder,and aqueous phases was sampled under pressure through the bottom diptube into another Hoke cylinder. Both phases were analyzed for glycolicacid concentration by HPLC.

For each initial glycolic acid concentration in the loaded solvent,Table 38 lists the final concentration of glycolic acid in each phase ofthe resulting mixture, and the partition coefficient for each initialglycolic acid concentration.

TABLE 38 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient loaded solvent aqueousphase organic phase (wt % org./wt % aq.) 3.9 2.8 1.35 0.48 11.3 6.2 2.30.37 16.0 9.4 3.2 0.34 17.5 10.2 3.6 0.35

Example 57 Back Extraction from a Loaded Solvent of Approximately 70%C8-C10 Trialkylamine in Combination with 10% Methyl Isobutyl Ketone and20% Kerosene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL aqueous solution of glycolic acid (20 wt % or 40wt %) and 10 mL of the loaded solvent (see Example 54). The vessel wasthen closed, and the headspace purged with nitrogen. The resultingmixture was stirred for 60 minutes at 140° C. under 40 psig (˜275.8 kPa)nitrogen. The stirring was stopped and the two phases allowed toseparate at 140° C., then the organic phase was sampled under pressurethrough the top dip tube into a Hoke cylinder, and aqueous phases wassampled under pressure through the bottom dip tube into another Hokecylinder. Both phases were analyzed for glycolic acid concentration byHPLC.

For each initial glycolic acid concentration in the loaded solvent andaqueous solution, Table 39 lists the final concentration of glycolicacid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 39 Initial wt % Initial wt % Final wt % Final wt % Partitionglycolic acid glycolic acid glycolic acid glycolic acid coefficient inaqueous in loaded in aqueous in organic (wt % org./wt phase solventphase phase % aq.) 20.0 16.0 21.9 11.7 0.54 40.0 17.5 34.4 18.7 0.54

Example 58 Back Extraction Using Water from a Loaded Solvent ofApproximately 70% C8-C10 Trialkylamine in Combination with 30% Toluene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL deionized water and 10 mL of the loaded solvent(see Example 54). The vessel was then closed, and the headspace purgedwith nitrogen. The resulting mixture was stirred for 60 minutes at 120°C. under 40 psig (˜275.8 kPa) nitrogen. The stirring was stopped and thetwo phases allowed to separate at 120° C., then the organic phase wassampled under pressure through the top dip tube into a Hoke cylinder,and aqueous phases was sampled under pressure through the bottom diptube into another Hoke cylinder. Both phases were analyzed for glycolicacid concentration by HPLC.

For each initial glycolic acid concentration in the loaded solvent,Table 40 lists the final concentration of glycolic acid in each phase ofthe resulting mixture, and the partition coefficient for each initialglycolic acid concentration.

TABLE 40 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient loaded solvent aqueousphase organic phase (wt % org./wt % aq.) 3.7 2.6 0.75 0.28 10.7 6.7 2.90.42 14.2 8.5 4.4 0.52 15.7 10.0 3.8 0.38

Example 59 Back Extraction from a Loaded Solvent of Approximately 70%C8-C10 Trialkylamine in Combination with 30% Toluene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL aqueous solution of glycolic acid (20 wt % or 40wt %) and 10 mL of the loaded solvent (see Example 54). The vessel wasthen closed, and the headspace purged with nitrogen. The resultingmixture was stirred for 60 minutes at 120° C. under 40 psig (˜275.8 kPa)nitrogen. The stirring was stopped and the two phases allowed toseparate at 120° C., then the organic phase was sampled under pressurethrough the top dip tube into a Hoke cylinder, and aqueous phases wassampled under pressure through the bottom dip tube into another Hokecylinder. Both phases were analyzed for glycolic acid concentration byHPLC.

For each initial glycolic acid concentration in the loaded solvent andaqueous solution, Table 41 lists the final concentration of glycolicacid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 41 Initial wt % Initial wt % Final wt % Final wt % Partitionglycolic acid glycolic acid glycolic acid glycolic acid coefficient inaqueous in loaded in aqueous in organic (wt % org./wt phase solventphase phase % aq.) 20.0 14.2 21.9 11.7 0.54 40.0 15.7 34.4 18.7 0.54

Example 60 Back Extraction Using Water from a Loaded Solvent ofApproximately 70% C8-C10 Trialkylamine in Combination with 30% Toluene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL deionized water and 10 mL of the loaded solvent(see Example 54). The vessel was then closed, and the headspace purgedwith nitrogen. The resulting mixture was stirred for 60 minutes at 140°C. under 40 psig (˜275.8 kPa) nitrogen. The stirring was stopped and thetwo phases allowed to separate at 140° C., then the organic phase wassampled under pressure through the top dip tube into a Hoke cylinder,and aqueous phases was sampled under pressure through the bottom diptube into another Hoke cylinder. Both phases were analyzed for glycolicacid concentration by HPLC.

For each initial glycolic acid concentration in the loaded solvent,Table 42 lists the final concentration of glycolic acid in each phase ofthe resulting mixture, and the partition coefficient for each initialglycolic acid concentration.

TABLE 42 Initial wt % Final wt % Final wt % glycolic acid in glycolicacid in glycolic acid in Partition coefficient loaded solvent aqueousphase organic phase (wt % org./wt % aq.) 3.7 2.7 0.70 0.26 10.7 7.6 2.30.30 14.2 9.5 3.6 0.38 15.7 10.1 2.8 0.28

Example 61 Back Extraction from a Loaded Solvent of Approximately 70%C8-C10 Trialkylamine in Combination with 30% Toluene

Into a 85-mL pressure reaction glass tube (pressure reaction vessel,from Andrews Glass Co.) equipped with magnetic stir bar and double diptubes was placed 10 mL aqueous solution of glycolic acid (20 wt % or 40wt %) and 10 mL of the loaded solvent (see Example 54). The vessel wasthen closed, and the headspace purged with nitrogen. The resultingmixture was stirred for 60 minutes at 140° C. under 40 psig (˜275.8 kPa)nitrogen. The stirring was stopped and the two phases allowed toseparate at 140° C., then the organic phase was sampled under pressurethrough the top dip tube into a Hoke cylinder, and aqueous phases wassampled under pressure through the bottom dip tube into another Hokecylinder. Both phases were analyzed for glycolic acid concentration byHPLC.

For each initial glycolic acid concentration in the loaded solvent andaqueous solution, Table 43 lists the final concentration of glycolicacid in each phase of the resulting mixture, and the partitioncoefficient for each initial glycolic acid concentration.

TABLE 43 Initial wt % Initial wt % Final wt % Final wt % Partitionglycolic acid glycolic acid glycolic acid glycolic acid coefficient inaqueous in loaded in aqueous in organic (wt % org./wt phase solventphase phase % aq.) 20.0 14.2 23.2 10.2 0.44 40.0 15.7 37.4 16.9 0.45

Example 62

Thermal Decomposition of Molten Ammonium Glycolate Salt Heated Up to˜133° C. for 3.5 Hours

Approximately 54.65 g of 25 wt % ammonium glycolate solution was addedto a 100-mL 3-neck flask, and water was removed by distillation. Whenthe weight of liquid in the flask decreased to 13.63 g, the clear,viscous liquid was analyzed: 12% (mol/mol) of ammonium glycolate wasconverted to glycolamide, and 13% (mol/mol) of ammonium glycolate wasconverted to glycolic dimer (as combined glycolic acid dimer andammonium glycolate dimer); glycolic acid (as combined glycolic acid andammonium glycolate) and ammonium recovery were 70% (mol/mol) and 73%(mol/mol) respectively (yields calculated based on final molarity ofproducts relative to final total molarity of ammonium glycolate andproducts). After a sample was removed for analysis by HPLC, IonElectrode, GC, etc. (referred to as “sampling” from here on), 12.06 g ofclear, viscous liquid remained in the flask.

A vacuum of 254 mm Hg was imposed, and heating continued up to 133° C.over a period of 3.5 hours. Analysis of the resulting product showed 24%(mol/mol) conversion of starting ammonium glycolate to glycolamide, 32%(mol/mol) conversion of starting ammonium glycolate to glycolic dimer(as combined glycolic acid dimer and ammonium glycolate dimer), 20%(mol/mol) glycolic acid recovery (as, combined glycolic acid andammonium glycolate) and 27% (mol/mol) of ammonium ion remaining. Thecombined yield of glycolic acid and glycolic acid dimer (separate fromthe ammonium glycolate and ammonium glycolate dimer present) wascalculated to be at least approximately 9%.

Example 63 Thermal Decomposition of Molten Ammonium Glycolate SaltHeated up to ˜140-150° C. for Approximately 6 Hours Followed byHydrolysis of Glycolic Acid Oligomers

Approximately 54.82 g of 25 wt % ammonium glycolate solution was addedto a 100-mL 3-neck flask, and water was removed by distillation. Whenthe weight of liquid in the flask decreased 21.15 g, about 8 (mol/mol) %ammonia had been removed. After sampling, 19.91 g of clear, viscousliquid was left in the flask. A vacuum up to 74 mm Hg was imposed, andthe temperature was raised to 140-150° C. within five hours andmaintained for about an hour. The weight of residual liquid in the flaskwas 10.33 g. Analysis of this product indicated 24% (mol/mol) ofammonium glycolate was converted to glycolamide, 28% (mol/mol) ofammonium glycolate was converted to glycolic dimer (as combined glycolicacid dimer and ammonium glycolate dimer); glycolic acid (as combinedglycolic acid and ammonium glycolate) and ammonium recovery were 20% and11% (mol/mol), respectively.

Approximately 7000 ppm of glycolide was also produced. After sampling, 9g of the liquid was mixed with 9 g of water. The resulting solution washeated to about 105° C. and refluxed for two hours. No further reductionin ammonium ion was observed, and dimer and oligomer were converted toglycolic acid. Glycolamide concentration did not change significantly.Yield of glycolic acid dimer (as combined glycolic acid dimer andammonium glycolate dimer) dropped from 24% (mol/mol) to 4% (mol/mol).Final glycolic acid recovery (as combined glycolic acid and ammoniumglycolate) was 56% (mol/mol). Yield of glycolic acid was at least 45%.

Example 64 Thermal Decomposition of Molten Ammonium Glycolate SaltHeated to ˜140-150° C. for Approximately 1 Hour Followed by Heating at170° C.

Approximately 54.85 g of 25 wt % ammonium glycolate solution was addedto a 100-mL 3-neck flask, and water was removed by distillation undervacuum (633 to 379 mm-Hg). When the weight of liquid in the flaskdecreased to 13.33 g, 10% (mol/mol) of ammonium glycolate was convertedto glycolamide, and 13% (mol/mol) of ammonium glycolate was converted toglycolic dimer (as combined glycolic acid dimer and ammonium glycolatedimer); glycolic acid (as combined glycolic acid and ammonium glycolate)and ammonium recovery were 72% and 76% (mol/mol) respectively. Aftersampling, 11.98 g of a clear, viscous liquid remained in the flask. Avacuum of 127 mm-Hg was imposed, and temperature was maintained at140-150° C. for an hour. Then the temperature was raised to 170° C. Theliquid color turned brown within minutes. Analysis of this productindicated 29% (mol/mol) of ammonium glycolate was converted toglycolamide, 16% (mol/mol) of ammonium glycolate was converted toglycolic dimer (as combined glycolic acid dimer and ammonium glycolatedimer); glycolic acid (as combined glycolic acid and ammonium glycolate)and ammonium ion recovery were 30% and 16% (mol/mol), respectively. Thecombined yield of glycolic acid and glycolic acid dimer was at leastapproximately 22%.

Example 65 Thermal Decomposition of Molten Ammonium Glycolate SaltHeated at 80° C. for Approximately 3 Hours Followed by Heating at 130°C. for 3 Hours

Approximately 29.1 g of 25% ammonium glycolate solution was producedfrom enzymatic hydrolysis of glycolonitrile using E. coli FM5/pNM18-H9cells (See U.S. 60/638,176; hereby incorporated by reference in itsentirety). The immobilized biocatalyst was decanted from the productsolution. The solution was then added to a 100-mL 3-neck flask, andwater was distilled off at 70-80° C. under vacuum (635-381 mm-Hg). Whenthe weight of liquid in the flask decreased to 7.02 g, 3% (mol/mol) ofammonium glycolate was converted to glycolamide, and 12% (mol/mol) ofammonium glycolate was converted to glycolic dimer (as combined glycolicacid dimer and ammonium glycolate dimer); glycolic acid (as combinedglycolic acid and ammonium glycolate) and ammonium recovery were 85% and76% (mol/mol) respectively After sampling, 4.2 g of a clear, viscousliquid remained in the flask. A vacuum of 127 mm-Hg was imposed and thetemperature was maintained at 80° C. for three hours, then at 130° C.for three hours. Analysis of this product showed 26 (mol/mol) %conversion from glycolate to glycolamide, 28% (mol/mol) conversion fromglycolate to glycolic dimer (as combined glycolic acid dimer andammonium glycolate dimer), 44% (mol/mol) glycolic acid (as combinedglycolic acid and ammonium glycolate) recovery, and 27% (mol/mol) ofammonium ion remained. Approximately 1.7 wt % of glycolide was alsoproduced. The combined yield of glycolic acid and glycolic acid dimerwas at least approximately 31

Example 66 Thermal Decomposition of Molten Ammonium Glycolate Salt(Lyophilized) Heated to Approximately 80-90° C. for 6 Hours

Approximately 341.7 g of 40 wt % ammonium glycolate solution was frozenand lyophilized to remove water. Then 146.4 g of lyophilized ammoniumglycolate was added to a flask, and heated to 80-90° C., then a vacuumof 50 mm-Hg was imposed. After six hours, no more ammonia was released.Final weight of the clear, viscous liquid was 104.4 g; 3% (mol/mol) ofammonium glycolate was converted to glycolamide, and 6% (mol/mol) ofammonium glycolate was converted to glycolic dimer (as combined glycolicacid dimer and ammonium glycolate dimer); glycolic acid (as combinedglycolic acid and ammonium glycolate) and ammonium recovery were 66%(mol/mol) and 62% (mol/mol), respectively. Approximately 0.14 wt % ofglycolide was also produced. The combined yield of glycolic acid andglycolic acid dimer was at least approximately 6%.

Example 67 Conversion of Glycolic Acid to Methyl Glycolate Using HeatedMethanol Vapor as Esterifying Agent and Stripping Gas

The purpose of Example 67 is to illustrate the ability of the presentprocess to convert an aqueous solution of glycolic acid into methylglycolate using heated methanol vapor as an esterifying agent andstripping gas. The methyl glycolate product was removed from thereaction chamber and selectively isolated from the vapor product streamusing a partial condenser.

¹H NMR Analytical Method Used in Examples 67-74

An aliquot of sample (0.40 mL) was mixed with an equal volume of CDCl₃containing 0.1% TMS (tetramethylsilane), and the resulting solutionanalyzed by ¹H NMR spectroscopy (500 MHz) and ¹³C NMR spectroscopy (125MHz). Samples were found to contain methanol, methyl glycolate, ammoniumglycolate and glycolic acid, and the ¹H NMR chemical shifts relative toTMS for the respective methylene and/or methoxy hydrogen atoms of thesecompounds are listed in Table 44 (when ammonium is not present insample) and Table 45 (when ammonium is present in the sample).

TABLE 44 Compound Reference Peaks Reference Peak CH₃OH CH ₃O—, singlet,δ = 3.388 HOCH₂C(O)OCH₃ HOCH ₂—, singlet, δ = 4.176 —OCH ₃, singlet, δ =3.758 HOCH₂C(O)OH HOCH ₂—, singlet, δ = 4.153When ammonium is present in the sample the glycolic acid peak shifts asfollows:

TABLE 45 Compound Reference Peaks Reference Peak CH₃OH CH ₃O—, singlet,δ = 3.388 HOCH₂C(O)OCH₃ HOCH ₂—, singlet, δ = 4.176 —OCH ₃, singlet, δ =3.758 HOCH₂C(O)OH HOCH ₂—, singlet, δ = 4.016 HOCH₂C(O)ONH₄ HOCH ₂—,singlet, δ = 3.945

The identity of the compounds identified in the table above wereconfirmed by individually adding methanol, methyl glycolate, ammoniumglycolate, or glycolic acid to a second aliquot of a reference sampleand observing an increase in the relative peak integration for therespective methylene and/or methoxy hydrogen atoms of these compounds.The molar ratios of the components of each sample were determined byintegration of the portion of the ¹H NMR spectra containing therespective methylene or methoxy H-atoms of the three chemicalcomponents.

Example 68 Conversion of Glycolic Acid to Methyl Glycolate Using HeatedMethanol Vapor as Esterifying Agent and Stripping Gas

The purpose of Example 68 is to illustrate the ability of the presentprocess to convert an aqueous solution of glycolic acid into methylglycolate using heated methanol vapor as an esterifying agent andstripping gas. The methyl glycolate product was removed from thereaction chamber and selectively isolated from the vapor product streamusing a partial condenser.

Approximately 137 g of polytetramethylene ether glycol (PTMEG, used as ahigh boiling point fluid; Lyondell PolyMeg® 1000, product number 9707;Lot #PEZM30B-A; Lyondell Chemical Company, Houston, Tex.;CAS#25190-06-1) was charged to the reaction chamber (300 cc autoclave).The pressure controller (loaded back-pressure regulator) was adjusted to25 psig (˜172.4 kPa). The autoclave agitator was started and set at 1000rpm. The autoclave internal temperature was set at 200° C. and the hotcondenser surface temperature was set at 130° C. Once temperaturesequilibrated, methanol (Brenntag Northeast Inc., Reading, Pa.; 99.99%pure, product code 838775) flow was initiated at 10 mL/min and methanolfeed temperature to the autoclave was maintained at 250° C. Theconditions were maintained for 45 minutes to allow the system to come toequilibrium. Glycolic acid feed (70 wt % aqueous solution;Sigma-Aldrich, Catalog #420581) was then initiates at 1.5 mL/min andmaintained for 45 minutes for a total feed of 67 mL. Methanol flow wascontinued an additional 20 minutes past termination of the ammoniumglycolate flow. Total methanol feed was 1160 mL.

Samples were collected from the hot condenser every 5 minutes during themethanol feed. The samples were analyzed by proton nuclear magneticresonance spectroscopy (¹H NMR) and found to contain methanol, methylglycolate, and a slight amount of glycolic acid. Results for samplesfrom the hot condenser (2-3, 2-5, and 2-7) and the stainless steelcollection drum (2-drum) are presented in Table 46. The molar ratio wasreported by standardizing the methyl glycolate peak (i.e. the “CH₃peak”) area to 1.

TABLE 46 Molar ratio of methanol, methyl glycolate, and glycolic acid insamples from the hot condenser or stainless steel collection drum. Molarratio calculated by standardizing methyl glycolate peak area to 1.Methyl Glycolic Collection time Sample Methanol Glycolate Acid (minutes)Identification No. (MeOH) (MeGLA) (GLA) 10-15 2-3 3.8 1.0 0.1 20-25 2-53.2 1.0 0.2 30-35 2-7 2.9 1.0 0.2 2-drum 199.5 1.0 nd nd—not detectable

The system was cooled down and samples were recovered from variousvessels and a material balance was performed. Total mass balanced within99%. The reactor contained 138 g of viscous liquid. The methanolrecovery drum contained 912 g. and the total weight of all samples was99 grams.

Example 69 Conversion of Ammonium Glycolate to Methyl Glycolate UsingHeated Methanol Vapor as Esterifying Agent and Stripping Gas (Reactortemperature ˜200° C.; Hot Condenser ˜130° C.)

The purpose of Example 69 is to show the direct conversion of an aqueoussolution of ammonium glycolate to methyl glycolate using heated methanolvapor as an esterifying agent and stripping gas.

An aqueous ammonium glycolate (NH₄GLA) solution (ammonium glycolate“solution A”) was prepared by combining 659 g of 70 wt % aqueousglycolic acid solution (Sigma-Aldrich) with 357 g of 30 wt % aqueousammonium hydroxide solution (EMD Chemicals, Darmstadt, Germany; productno. AX1303-6).

Approximately 138 g of PTMEG was charged to the reactor (autoclave). Thepressure controller was adjusted to 25 psig (˜172.4 kPa). The autoclaveagitator was started and set at 1000 rpm. The autoclave temperature wasset at 200° C. and the hot condenser was set at 130° C. Oncetemperatures equilibrated, methanol flow was initiated at 10 mL/min andmethanol feed temperature to the autoclave was maintained at 250° C. Theconditions were maintained for 15 minutes to allow the system to come toequilibrium. Ammonium Glycolate solution A was then pumped to thereactor at a rate of 2.2 mL/min and maintained for 60 minutes for atotal feed of 132 mL. Methanol flow was continued an additional 35minutes past termination of the ammonium glycolate feed. Total methanolfeed was 1110 mL.

Samples were collected from the hot condenser every 5 minutes during theammonium glycolate feed. The first 30 minutes of samples were combinedto make samples designated with an “A” and the second 30 minutes ofsamples were combined to make samples designated with a “B”. The samples(samples “5A” and “5B”) were analyzed by ¹H NMR and found to containmethanol, methyl glycolate, and ammonium glycolate. Results aresummarized in Table 47

The system was cooled down and samples were recovered from variousvessels and a material balance was performed. The autoclave contained140 g of viscous liquid. The methanol recovery drum contained 913 g andthe total weight of all samples was 123 grams.

Example 70 Conversion of Ammonium Glycolate to Methyl Glycolate UsingHeated Methanol Vapor as Esterifying Agent and Stripping Gas (ReactorTemperature ˜170° C.; Hot Condenser ˜100° C.)

Equipment and procedures were identical to Example 69 except the reactor(autoclave) temperature was maintained at 170° C. and the hot condenserwas maintained at 100° C. Ammonium glycolate solution A was fed for 60minutes and samples were combined as described in Example 68 to preparesamples “7A” and “7B”. Results are summarized in Table 47.

Example 71 Conversion of Ammonium Glycolate to Methyl Glycolate UsingHeated Methanol Vapor as Esterifying Agent and Stripping Gas (MineralOil as Heat Transfer Fluid; Reactor ˜170° C.; Hot Condenser ˜100° C.)

Equipment and procedures were identical to those described in Example 70unless otherwise noted.

Approximately 131 g of mineral oil (MultiTherm PG-1® heat transferfluid, MultiTherm® LLC, Malvern, Pa.) was added to the reactor. Theautoclave temperature was maintained at 170° C. and the hot condenserwas maintained at 100° C. Ammonium glycolate solution A was fed for 60minutes and samples were combined like Example 69 to prepare samples“8A” and “8B”. Results are summarized in Table 47.

Example 72 Conversion of Ammonium Glycolate to Methyl Glycolate UsingHeated Methanol Vapor as Esterifying Agent and Stripping Gas (MineralOil as Heat Transfer Fluid; Reactor ˜200° C.; Hot Condenser ˜130° C.)

Equipment and procedures were identical to those described in Example 69unless otherwise noted.

Approximately 128 g of mineral oil (MultiTherm PG-1® heat transferfluid, MultiTherm® LLC, Malvern, Pa.) was added to the reactor. Theautoclave temperature was maintained at 200° C. and the hot condenserwas maintained at 130° C. An ammonium glycolate solution (ammoniumglycolate “solution B”) was prepared by combining 75 g of glycolic acidcrystals (99% glycolic acid, Sigma Aldrich Catalogue #124737) with 68.5g of aqueous ammonium hydroxide solution (30 wt %, EMD Chemicals) and 25g of deionized water. Ammonium glycolate solution B was fed for 60minutes and samples were combined like Example 69 to prepare samples“9A” and “9B”. Results are summarized in Table 47.

Example 73 Conversion of Ammonium Glycolate to Methyl Glycolate UsingHeated Methanol Vapor as Esterifying Agent and Stripping Gas (No highboiling point Fluid; Reactor 200° C.; Hot Condenser ˜130° C.)

Equipment and procedures were identical to Example 69 except no highboiling point fluid was used. Instead, the agitator was removed and 94grams of packing material (“ProPak” ¼ inch high efficiency packing madefrom Hastelloy® C276, Ace Glass Inc.) was added to the reactor(autoclave). The methanol feed line was inserted through the packing somethanol addition was at the bottom of the autoclave. An ammoniumglycolate solution (ammonium glycolate “solution C”) was prepared bycombining equal mass of 70 wt % aqueous glycolic acid solution (SigmaAldrich) and 30 wt % aqueous ammonium hydroxide solution (EMD Chemicals)followed by minor adjustments with GLA and ammonium to achieve a pHbetween 7.0 and 7.5. The ammonium glycolate feed was added to the top ofthe packing in the reactor.

The reactor temperature was maintained at 200° C. and the hot condenserwas maintained at 130° C. Ammonium glycolate solution C was fed at 2.2mL/minute for 60 minutes and samples were combined like Example 69 toprepare samples “11A” and “11B”. Results are summarized in Table 47.

Example 74 Conversion of Ammonium Glycolate to Methyl Glycolate UsingHeated Methanol Vapor as Esterifying Agent and Stripping Gas (No HeatTransfer Fluid; Reactor ˜170° C.; Hot Condenser ˜100° C.)

Equipment and procedures were identical to Example 73 except theautoclave temperature was maintained at 170° C. and the hot condenserwas maintained at 100° C. Ammonium glycolate “solution C” was fed for 60minutes and samples were combined like Example 69 to prepare samples“13A” and “13B”. Results are summarized in Table 47.

TABLE 47 Molar ratio of methanol, methyl glycolate, ammonium glycolate,and glycolic acid in samples from the hot condenser. Molar ratiocalculated by standardizing methyl glycolate peak area to 1. SampleCollection Identi- Methyl Glycolic Ammonium Time fication MethanolGlycolate Acid Glycolate (minutes) No. (MeOH) (MeGLA) (GLA) (NH₄GLA) 0-30 5A 17.4 1.0 0.66 0.19 30-60 5B 6.7 1.0 0.45 0.09  0-30 7A 26.8 1.00.30 0.15 30-60 7B 8.8 1.0 0.24 0.13  0-30 8A 11.7 1.0 0.21 0.15 30-608B 52.7 1.0 0.47 0.51  0-25 9A 12.8 1.0 0.49 0.15 25-50 9B 6.1 1.0 0.470.17  0-30 11A  70.6 1.0 1.18 0.24 30-60 11B  26.6 1.0 0.59 0.16  0-3013A  27.4 1.0 0.26 0.17 30-60 13B  17.9 1.0 0.19 0.17

1. A process for producing glycolic acid from formaldehyde and hydrogencyanide comprising: (a) providing an aqueous formaldehyde feed streamthat is heated to a temperature of about 90° C. to about 150° C. for adeterminable period of time; (b) contacting the heated aqueous feedstream of (a) with hydrogen cyanide at a temperature suitable forglycolonitrile synthesis, whereby glycolonitrile is produced; (c)contacting the glycolonitrile of step (b) in a suitable aqueous reactionmixture with an enzyme catalyst comprising a polypeptide havingnitrilase activity, said polypeptide having the amino acid sequence ofSEQ ID NO:30, whereby glycolic acid is produced; and (d) recovering theglycolic acid produced in (c) in the form of a salt or acid.
 2. Theprocess of claim 1 wherein the glycolic acid is recovered using arecovery method selected from the group consisting of reactive solventextraction, ion exchange, electrodialysis, polymerization, thermaldecomposition, alcoholysis, and combinations thereof.
 3. The process ofclaim 2 wherein the recovery method is selected from the groupconsisting of ion exchange and reactive solvent extraction.
 4. Theprocess of claim 1 wherein an amount of sodium hydroxide is added to theaqueous formaldehyde feed stream prior to heating the aqueousformaldehyde feed stream wherein the molar ratio of sodium hydroxide toformaldehyde is about 1:50 to about 1:2000.
 5. The process of claim 1wherein the molar ratio of hydrogen cyanide to formaldehyde is at least1.01:1 to about 1.15:1.
 6. The process of claim 1 wherein the heatedaqueous formaldehyde feed stream is reacted with hydrogen cyanide at areaction temperature of about 0° C. to about 70° C.
 7. The process ofclaim 6 wherein the heated aqueous formaldehyde feed stream is reactedwith hydrogen cyanide at a reaction temperature of about 10° C. to about30° C.
 8. The process of claim 1 wherein the aqueous formaldehyde feedstream comprises about 0.1 wt to about 15 wt methanol.
 9. The process ofclaim 1 wherein the enzyme catalyst is in the form of a whole microbialcell, a permeabilized microbial cell, a microbial cell extract,partially purified enzyme, or purified enzyme.
 10. The process of claim9 wherein said whole microbial cell is a transformed microbial host cellrecombinantly expressing said polypeptide.
 11. The process of claim 10wherein the transformed microbial host cell is selected from the groupconsisting of Comamonas sp., Corynebacterium sp., Brevibacterium sp.,Rhodococcus sp., Azotobacter sp., Citrobacter sp., Enterobacter sp.,Clostridium sp., Klebsiella sp., Salmonella sp., Lactobacillus sp.,Aspergillus sp., Saccharomyces sp., Zygosaccharomyces sp., Pichia sp.,Kluyveromyces sp., Candida sp., Hansenula sp., Dunaliella sp.,Debaryomyces sp., Mucor sp., Torulopsis sp., Methylobacteria sp.,Bacillus sp., Escherichia sp., Pseudomonas sp., Rhizobium sp., andStreptomyces sp.
 12. The process of claim 11 wherein the transformedmicrobial host is Escherichia coli.
 13. The process of claim 12 whereinthe transformed microbial host cell is an Escherichia coli strainselected from the group consisting of E. coli MG1655 havinginternational depository number ATCC 47076 and E. coli FM5 havinginternational depository number ATCC
 53911. 14. The process of any oneof claims 9-13 wherein the enzyme catalyst is immobilized in or on asoluble or insoluble support.
 15. The process of claim 1 wherein theconcentration of ammonium glycolate produced in the aqueous reactionmixture is about 0.02 wt % to about 90 wt %.
 16. The process of claim 15wherein the concentration of ammonium glycolate produced in the aqueousreaction mixture is about 0.02 wt % to about 40 wt %.
 17. The process ofclaim 1 wherein the glycolonitrile concentration in the aqueous reactionmixture is in the range of about 5 mM to about 1 M.
 18. The process ofclaim 17 wherein the glycolonitrile concentration in the aqueousreaction mixture is maintained by continuous or aliquot addition. 19.The process of claim 1 wherein the pH in the aqueous reaction mixture ismaintained between about 5.5 and about 7.7.
 20. The process of claim 1wherein the enzymatic conversion of glycolonitrile to glycolic acidoccurs under substantially oxygen free conditions.
 21. The process ofclaim 1 wherein the aqueous reaction mixture further comprises astabilizer selected from the group consisting of potassium thiosulfateand sodium dithionite at a concentration of less than 5 wt %.
 22. Theprocess of claim 1, wherein said enzyme catalyst provides a catalystproductivity of at least 300 grams of glycolic acid per gram dry cellweight of enzyme catalyst.
 23. The process of claim 22, wherein saidenzyme catalyst provides a catalyst productivity of at least 450 gramsof glycolic acid per gram dry cell weight of enzyme catalyst.
 24. Theprocess of claim 23, wherein said enzyme catalyst provides a catalystproductivity of at least 1000 grams of glycolic acid per gram dry cellweight of enzyme catalyst.
 25. A process for producing glycolic acidfrom formaldehyde and hydrogen cyanide comprising: (a) providing anaqueous formaldehyde feed stream that is heated to a temperature ofabout 90° C. to about 150° C. for a determinable period of time; (b)contacting the heated aqueous feed stream of (a) with hydrogen cyanideat a temperature suitable for glycolonitrile synthesis, wherebyglycolonitrile is produced; (c) contacting the glycolonitrile of step(b) in a suitable aqueous reaction mixture with an enzyme catalystcomprising a polypeptide having nitrilase activity, said polypeptidehaving the amino acid sequence of SEQ ID NO:30, whereby glycolic acid isproduced; and (d) recovering the glycolic acid produced in (c) by ionexchange; wherein said glycolic acid has a purity of at least 99.9%.